Induction of Salmonella Enteritidis into a Viable but Nonculturable State by Cinnamaldehyde and Its Resuscitation.

Trans-cinnamaldehyde (TC), a typical plant-derived compound, has been widely used in the control of foodborne pathogen contamination. Nevertheless, the risk associated with the occurrence of viable but nonculturable (VBNC) bacteria induced by TC remains unclear. The results of this study showed that Salmonella Enteritidis (S. Enteritidis) entered the VBNC state after being induced by TC at a minimum inhibitory concentration of 312.5 µg/mL and survived for at least 22 days under TC treatment. Enhanced resistance was found against heat treatment (75°C, 30 s), antibiotics (i.e., ampicillin, ceftriaxone sodium, chloramphenicol), and hydrogen peroxide (3%) in VBNC S. Enteritidis. A synergistic effect against VBNC S. Enteritidis occurred when TC was combined with acid treatment, including lactic acid and acetic acid (pH = 3.5). VBNC and resuscitated S. Enteritidis by sodium pyruvate treatment (100 mM) were found to retain the infectious ability to Caco-2 cells. Relative expression levels of the stress-related genes relA, spoT, ppx, lon, katG, sodA, dnaK, and grpE were upregulated in VBNC S. Enteritidis. Accumulation of reactive oxygen species (ROS) and protein aggregates was observed in VBNC cells. Besides, the resuscitation of VBNC cells was accompanied with clearance of ROS and protein aggregates. In summary, this study presents a comprehensive characterization of stress tolerance and resuscitation of VBNC S. Enteritidis induced by cinnamaldehyde, and the results provide useful information for the development of effective control strategy against VBNC pathogenic bacteria in food production.

Stress responses and Physiological Changes of Salmonella enterica Serovar Enteritidis on Short-Term and Long-Term Benzalkonium Bromide Adaptation.

Salmonella enterica is a common foodborne pathogen that poses significant safety risks across the world. And benzalkonium bromide (BK) is widely used as a disinfectant to sterilize the food processing equipment. It has been reported that sub-lethal concentration of disinfectants induced not only the homologous resistance but also cross-resistances. This work analyzed the induced resistances of Salmonella Enteritidis by short-term adaptation (STA) and long-term adaptation (LTA) to BK. We have demonstrated that inefficient sterilization exposes Salmonella Enteritidis to sub-lethal concentrations of BK, and adapts bacteria to a higher minimum inhibitory concentration and minimum bactericidal concentration. In addition, STA, but not LTA, to BK induced heterogeneous resistance to sodium hypochlorite, and cross-resistance to freezing, desiccation, and heating, which may be caused by the membrane composition change of Salmonella Enteritidis. This work could be useful to the optimization of cleaning protocol.

Does internet use benefit the mental health of older adults? Empirical evidence from the China health and retirement longitudinal study.

The mental health (MH) of older adults is a prominent public health concern. However, research regarding the impact of emerging Internet use on MH among older adults remains limited, particularly in transitional economies experiencing a rapidly aging population such as China. Thus, to address this research gap, this study uses data from the 2013-2018 waves of the China Health and Retirement Longitudinal Study. To investigate the causal relationship between Internet use and MH among older adults and explore the underlying channels through which this relationship operates. The results reveal a notable positive association between Internet use and MH among older adults. Furthermore, the study highlights social interaction, social trust, traveling expenses, and healthy habits as crucial channels through which Internet use can impact MH among older adults. The analysis also reveals how Internet use demonstrates a stronger positive effect on older individuals who have fewer chronic diseases and live with their offspring compared with their counterparts. These findings have significant policy implications, which thus emphasizes the need to enhance Internet use among older adults as a means of improving their MH.

Genomic epidemiology of hypervirulent Listeria monocytogenes CC619: Population structure, phylodynamics and virulence.

Listeria monocytogenes is a ubiquitous foodborne pathogen causing human and animal listeriosis with high mortality. Neurological and maternal-neonatal listeriosis outbreaks in Europe and the United States were frequently associated with clonal complexes CC1, CC2 and CC6 harboring Listeria Pathogenicity Island-1 (LIPI-1), as well as CC4 carrying both LIPI-1 and LIPI-4. However, human listeriosis in China was predominantly linked to CC87 and CC619 from serotype 1/2b. To understand the genetic evolution and distribution patterns of CC619, we characterized the epidemic history, population structure, and transmission feature of CC619 strains through analysis of 49,421 L. monocytogenes genomes globally. We found that CC619 was uniquely distributed in China, and closely related with perinatal infection. As CC619 strains were being mainly isolated from livestock and poultry products, we hypothesized that pigs and live chicken were the reservoirs of CC619. Importantly, all CC619 strains not only harbored the intact LIPI-1 and LIPI-4, but these also carried LIPI-3 that could facilitate host colonization and invasion. The deficiency of LIPI-3 or LIPI-4 markedly decreased L. monocytogenes colonization capacity in a model of intragastric infection in the mouse. Altogether, our findings suggest that the hypervirulent CC619 harboring three pathogenicity islands LIPI-1, LIPI-3 and LIPI-4 is a putatively persistent population in various foods, environment, and human population, warranting the further research for deciphering its pathogenicity and strengthening epidemiological surveillance.

Effect of RpoS on the survival, induction, resuscitation, morphology, and gene expression of viable but non-culturable Salmonella Enteritidis in powdered infant formula.

Involvement of the transcriptional regulator RpoS in the persistence of viable but non-culturable (VBNC) state has been demonstrated in several species of bacteria. This study investigated the role of the RpoS in the formation and resuscitation of VBNC state in Salmonella enterica serovar Enteritidis CICC 21482 by measuring bacterial survival, morphology, physiological characteristics, and gene expression in wild-type (WT) and rpoS-deletion (ΔrpoS) strains during long-term storage in powdered infant formula (PIF). The ΔrpoS strain was produced by allelic exchange using a suicide plasmid. Bacteria were inoculated into PIF for 635-day storage. Survival, morphology, intracellular reactive oxygen species (ROS) levels and intercellular quorum sensing autoinducer-2 (AI-2) contents were regularly measured. Resuscitation assays were conducted after obtaining VBNC cells. Gene expression was measured using real-time quantitative polymerase chain reaction (qPCR). The results showed that RpoS and low temperature conditions were associated with enhanced culturability and recoverability of Salmonella Enteritidis after desiccation storage in low water activity (aw) PIF. In addition, the synthesis of intracellular ROS and intercellular quorum sensing AI-2 was regulated by RpoS, inducing the formation and resuscitation of VBNC cells. Gene expression of soxS, katG and relA was found strongly associated with RpoS. Due to the lack of RpoS factor, the ΔrpoS strain could not normally synthesize SoxS, catalase and (p)ppGpp, resulting in its early shift to the VBNC state. This study elucidates the role of rpoS in desiccation stress and the formation and resuscitation mechanism of VBNC cells under desiccation stress. It serves as the basis for preventing and controlling the recovery of pathogenic bacteria in VBNC state in low aw foods.

Heat Resistance, Virulence, and Gene Expression of Desiccation-Adapted Salmonella Enteritidis During Long-Term Storage in Low-Water Activity Foods.

Desiccation stress could induce crossprotection and even affect virulence of Salmonella enterica. However, the influence of food matrices with low-water activity on desiccation adaptation of Salmonella still remains unclear. This study investigated the survival and adaptation of Salmonella Enteritidis in skim milk powder, ginger powder, and chocolate powder under desiccation storage conditions for a total of 12 weeks. High survival rates of Salmonella Enteritidis in all food matrices maintained over the long-term desiccation storage. Desiccation-adapted Salmonella Enteritidis enhanced heat resistance (p < 0.05) with the increase of storage time. Food composition plays an important role in the induction of crossresistance of desiccation-adapted Salmonella. After desiccation storage, Salmonella Enteritidis in ginger powder was most tolerant to heat treatment. Salmonella Enteritidis in skim milk powder was most resistant to the gastrointestinal simulation environment, and had strongest adhesion to Caco-2 cells. The effects of food composition on gene expression (rpoS, proV, otsA, otsB, grpE, dnaK, rpoH, and sigDE) in desiccation-adapted Salmonella Enteritidis were not significant (p > 0.05). At initial desiccation storage, osmotic protection-related genes (fadA, proV, otsA, and otsB), stress response regulator (rpoS), and heat-resistance-related genes (grpE, dnaK, and rpoH) were all significantly upregulated (p < 0.05). However, after 4-week storage, the expression level of desiccation-related genes, proV, otsA, otsB, grpE, dnaK, and rpoH, significantly decreased (p < 0.05). This study enables a better understanding of Salmonella's responses to long-term desiccation stress in different kinds of low-water activity foods.

Is the happiness of Chinese truly the highest in the world? The impact of basic public services on happiness.

Based on the survey report by the United Nations Sustainable Development Solutions Network (SDSN) and Ipsos Group, the world ranking of Chinese people's happiness shows a significant gap. This study attempts to analyze the subjective well-being of Chinese residents through public database from the China Household Finance Survey Center in 2017. An ordered Probit model is constructed to investigate the impact of non-monetary factors, specifically basic public services, on the subjective well-being of Chinese people. The results indicate that: (1) The subjective well-being of Chinese residents is found to be lower than what the survey report indicated. (2) Basic public services have a significant positive impact on residents' happiness. (3) Social trust played a moderating role, positively influencing the relationship between basic public services and residents' happiness. (4) The impact of basic public services on happiness varied significantly depending on factors such as age, registered residence, and places of residence. To enhance the happiness of Chinese residents, it is recommended to focus on improving the equalization of basic public services and establishing a robust basic public service system. These measures can effectively contribute to the overall well-being and happiness of the population.

Prevalence and transmission risk of colistin and multidrug resistance in long-distance coastal aquaculture.

Due to the wide use of antibiotics, intensive aquaculture farms have been recognized as a significant reservoir of antibiotic resistomes. Although the prevalence of colistin resistance genes and multidrug-resistant bacteria (MDRB) has been documented, empirical evidence for the transmission of colistin and multidrug resistance between bacterial communities in aquaculture farms through horizontal gene transfer (HGT) is lacking. Here, we report the prevalence and transmission risk of colistin and multidrug resistance in 27 aquaculture water samples from 9 aquaculture zones from over 5000 km of subtropical coastlines in southern China. The colistin resistance gene mcr-1, mobile genetic element (MGE) intl1 and 13 typical antibiotic resistance genes (ARGs) were prevalent in all the aquaculture water samples. Most types of antibiotic (especially colistin) resistance are transmissible in bacterial communities based on evidence from laboratory conjugation and transformation experiments. Diverse MDRB were detected in most of the aquaculture water samples, and a strain with high-level colistin resistance, named Ralstonia pickettii MCR, was isolated. The risk of horizontal transfer of the colistin resistance of R. pickettii MCR through conjugation and transformation was low, but the colistin resistance could be steadily transmitted to offspring through vertical transfer. The findings have important implications for the future regulation of antibiotic use in aquaculture farms globally to address the growing threat posed by antibiotic resistance to human health.

Comparative genomics unveils extensive genomic variation between populations of Listeria species in natural and food-associated environments.

Comprehending bacterial genomic variation linked to distinct environments can yield novel insights into mechanisms underlying differential adaptation and transmission of microbes across environments. Gaining such insights is particularly crucial for pathogens as it benefits public health surveillance. However, the understanding of bacterial genomic variation is limited by a scarcity of investigations in genomic variation coupled with different ecological contexts. To address this limitation, we focused on Listeria, an important bacterial genus for food safety that includes the human pathogen L. monocytogenes, and analyzed a large-scale genomic dataset collected by us from natural and food-associated environments across the United States. Through comparative genomics analyses on 449 isolates from the soil and 390 isolates from agricultural water and produce processing facilities representing L. monocytogenes, L. seeligeri, L. innocua, and L. welshimeri, we find that the genomic profiles strongly differ by environments within each species. This is supported by the environment-associated subclades and differential presence of plasmids, stress islands, and accessory genes involved in cell envelope biogenesis and carbohydrate transport and metabolism. Core genomes of Listeria species are also strongly associated with environments and can accurately predict isolation sources at the lineage level in L. monocytogenes using machine learning. We find that the large environment-associated genomic variation in Listeria appears to be jointly driven by soil property, climate, land use, and accompanying bacterial species, chiefly representing Actinobacteria and Proteobacteria. Collectively, our data suggest that populations of Listeria species have genetically adapted to different environments, which may limit their transmission from natural to food-associated environments.

Metabolomics-based response of Salmonella to desiccation stress and skimmed milk powder storage.

The strong survival ability of Salmonella in low-moisture foods (LMFs) has been of public concern, and is considered a threat to people's health. Recently, the development of omics technology has promoted research on the molecular mechanisms of the desiccation stress response of pathogenic bacteria. However, multiple analytical aspects related to their physiological characteristics remain unclear. We explored the physiological metabolism changes of S. enterica Enteritidis exposed to a 24 h-desiccation treatment and a subsequent 3-month desiccation storage in skimmed milk powder (SMP) with an approach of gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography-Q Exactive-mass spectrometry (UPLC-QE-MS). A total of 8,292 peaks were extracted, of which 381 were detected by GC-MS and 7,911 peaks were identified by LC-MS/MS, respectively. Through analyses of differentially expressed metabolites (DEMs) and key pathways, a total of 58 DEMs emerged from the 24 h-desiccation treatment, which exhibited the highest relevance for five metabolic pathways, involving glycine, serine, and threonine metabolism, pyrimidine metabolism, purine metabolism, vitamin B6 metabolism, and pentose phosphate pathway. After 3-month SMP storage, 120 DEMs were identified, which were related to several regulatory pathways including arginine and proline metabolism, serine and threonine metabolism, ß-alanine metabolism, glycerolipid metabolism, and glycolysis. The analyses of key enzyme activities of XOD, PK, and G6PDH and ATP content provided further evidence that supported the metabolic responses such as nucleic acid degradation, glycolysis, and ATP production played an important role in Salmonella's adaptation to desiccation stress. This study enables a better understanding of metabolomics-based responses of Salmonella at the initial stage of desiccation stress and the following long-term adaptive stage. Meanwhile, the identified discriminative metabolic pathways may serve as potentially useful targets in developing strategies for the control and prevention of desiccation-adapted Salmonella in LMFs.

Effect of honey dressing in the management of diabetic foot ulcers: A meta-analysis.

A meta-analysis study to assess the effect of honey dressing (HD) in the management of diabetic foot ulcer (DFU). A comprehensive literature examination till January 2023 was implemented and 1794 linked studies were appraised. The picked studies contained 882 subjects with DFUs were in the picked studies' baseline, 424 of them were using HD, and 458 were using a control. Odds ratio (OR) in addition to 95% confidence intervals (CIs) were used to calculate the consequence of HD in the management of DFUs after DFU by the dichotomous and continuous styles and a fixed or random model. The HD applied to DFUs caused a significantly higher wound healing rate (OR, 2.06; 95% CI, 1.45-2.93, P < .001) and lower wound healing time (MD, -10.42; 95% CI, -16.27- -4.58, P < .001) compared with the control. The HD applied to DFUs caused a significantly higher wound healing rate and lower wound healing time compared with the control. Although precautions should be taken when commerce with the consequences since most of the picked studies for this meta-analysis was with low sample sizes.

Gasdermin A Is Required for Epidermal Cornification during Skin Barrier Regeneration and in an Atopic Dermatitis-Like Model.

Atopic dermatitis is featured with impaired skin barrier. The stratum corneum and the intercellular tight junctions constitute the permeability barrier, which is essential to protect water loss in the host and prevent pathogen entry. The epidermal barrier is constantly renewed by differentiating keratinocytes through cornification, during which autophagy contributes to elimination of organelles and nucleus. The human GSDMA and its mouse homologs Gsdma1-3 are expressed in the suprabasal epidermis. Although a pyroptotic role of GSDMA/Gsdma1 in host defense against Streptococcus pyogenes has been reported, the physiological function of Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Here, through repeated epidermal barrier disruption, we found that tight junction formation and stratum corneum maturation were defective in the Gsdma1/a3-deficient epidermis. Using comparative gene profiling analysis, mitochondrial respiration measurement, and in vivo tracing of mitophagy, our data indicate that Gsdma1/a3 activation leads to mitochondrial dysfunction and subsequently facilitates mitochondrial turnover and epidermal cornification. In calcipotriol (MC903)-induced atopic dermatitis-like animal model, we showed that Gsdma1/a3-deficiency selectively enhanced the T helper type 2 response. Remarkably, the GSDMA expression is reduced in the epidermis of patients with atopic dermatitis compared with that of normal individuals. Gsdma1/a3-deficiency might be involved in atopic dermatitis pathogenesis, likely through GSDMA-mediated epidermal differentiation and cornification.

Exploring Cantharidin and its Analogues as Anticancer Agents: A Review.

BACKGROUND: Cantharidin (CTD) is a highly toxic substance which can be used to treat a variety of cancers. However, the clinical application of CTD is restricted due to the serious side effects. In recent years, screening its analogues, exploring the mechanism of action and using combinatory therapy with certain substances are considered to be feasible methods which can reduce side effects and improve the therapeutic activity of CTD. This review aims to describe SAR (structure-activity relationship) of CTD analogues, CTD induction mechanisms, and combinatory therapy exploration. METHODS: We searched for research about CTD by entering the database. Important information was screened and extracted purposefully, including SAR, mechanisms, methods, etc. Finally, these contents were unified into a framework to form a review. RESULTS: Some CTD analogues with imidazolium salt or double bonds at C-5 and C-6 positions demonstrate good anticancer activity. Through introducing methyl and acetoxy groups at the C-1 or C-4 position, the inhibitory effect of PP was weakened or even inactivated. Removing the two methyl groups of C-2 and C-3 can reduce side effects and improve efficacy. Replacing methyl with fluorine can also improve the activity and reduce toxicity. Water solubility and bioavailability could be improved by opening the five fivemembered anhydride ring to form carboxylic acid, salt, amide, and ester derivatives. The anticancer mechanism can be divided into the following aspects, including inhibiting cell invasion and metastasis, inducing apoptosis, regulating cell cycle and enhancing immunity. The proper formulation of CTD and its analogues (liposomes, nanoparticles and micelles) can improve the targeting of liver cancer and reduce toxic and side effects. CTD combined with anti-angiogenic therapeutics (Ginsenoside Rg3, Bevacizumab, Apatinib and Endostar) showed additive anti-pancreatic cancer effects. CONCLUSION: It was found that the potential mechanism was closely related to multi-channel and multi-target interactions, which provided a guiding direction for the later exploration of new clinical therapeutic applications. However, some detailed mechanisms are still unclear, and more evidence is required to verify. In addition, the new methods to improve the therapeutic potential of CTD and its analogues still need more clinical trials to be tested in the future. This prospect is very broad and worthy of further study.

Genomic characterization of Salmonella isolated from retail chicken and humans with diarrhea in Qingdao, China.

Salmonella, especially antimicrobial resistant strains, remains one of the leading causes of foodborne bacterial disease. Retail chicken is a major source of human salmonellosis. Here, we investigated the prevalence, antimicrobial resistance (AMR), and genomic characteristics of Salmonella in 88 out of 360 (24.4%) chilled chicken carcasses, together with 86 Salmonella from humans with diarrhea in Qingdao, China in 2020. The most common serotypes were Enteritidis and Typhimurium (including the serotype I 4,[5],12:i:-) among Salmonella from both chicken and humans. The sequence types were consistent with serotypes, with ST11, ST34 and ST19 the most dominantly identified. Resistance to nalidixic acid, ampicillin, tetracycline and chloramphenicol were the top four detected in Salmonella from both chicken and human sources. High multi-drug resistance (MDR) and resistance to third-generation cephalosporins resistance were found in Salmonella from chicken (53.4%) and humans (75.6%). In total, 149 of 174 (85.6%) Salmonella isolates could be categorized into 60 known SNP clusters, with 8 SNP clusters detected in both sources. Furthermore, high prevalence of plasmid replicons and prophages were observed among the studied isolates. A total of 79 antimicrobial resistant genes (ARGs) were found, with aac(6')-Iaa, blaTEM-1B, tet(A), aph(6)-Id, aph(3″)-Ib, sul2, floR and qnrS1 being the dominant ARGs. Moreover, nine CTX-M-type ESBL genes and the genes blaNMD-1, mcr-1.1, and mcr-9.1 were detected. The high incidence of MDR Salmonella, especially possessing lots of mobile genetic elements (MGEs) in this study posed a severe risk to food safety and public health, highlighting the importance of improving food hygiene measures to reduce the contamination and transmission of this bacterium. Overall, it is essential to continue monitoring the Salmonella serotypes, implement the necessary prevention and strategic control plans, and conduct an epidemiological surveillance system based on whole-genome sequencing.

LncRNA DUXAP8 induces breast cancer radioresistance by modulating the PI3K/AKT/mTOR pathway and the EZH2-E-cadherin/RHOB pathway.

Radiation resistance poses a major clinical challenge in breast cancer (BC) treatment, but little is known about how long noncoding RNA (lncRNA) may regulate this phenomenon. Here, we reported that DUXAP8 was highly expressed in radioresistant BC tissues, and high expression of DUXAP8 was associated with poor prognosis. We found that the overexpression of DUXAP8 promoted radioresistance, while the knockdown of DUXAP8 expression increased radiosensitivity. Further studies revealed that DUXAP8 enhanced the radioresistance of BC cells by activating the PI3K/AKT/mTOR pathway and by repressing the expression of E-cadherin and RHOB through interaction with EZH2. Together, our work demonstrates that the overexpression of DUXAP8 promotes the resistance of BC cells toward radiation through modulating PI3K/AKT/mTOR pathway and EZH2-E-cadherin/RHOB axis. Targeting DUXAP8 may serve as a potential strategy to overcome radioresistance in BC treatment.

Draft Genome Sequences of Antibiotic-Resistant Serratia sp. Strains Isolated from Raw Sewage and Sediment Sludge in Georgia, USA.

Sewage has been established as a prime matrix for monitoring the emergence and dissemination of etiologic agents and antibiotic resistance determinants in a population. Here, we report the draft genomes and the acquired resistance genes of 11 antibiotic-resistant Serratia sp. isolates that were detected in samples from wastewater treatment plants.

Integrative Assessment of Reduced Listeria monocytogenes Susceptibility to Benzalkonium Chloride in Produce Processing Environments.

For decades, quaternary ammonium compounds (QAC)-based sanitizers have been broadly used in food processing environments to control foodborne pathogens such as Listeria monocytogenes. Still, there is a lack of consensus on the likelihood and implication of reduced Listeria susceptibility to benzalkonium chloride (BC) that may emerge due to sublethal exposure to the sanitizers in food processing environments. With a focus on fresh produce processing, we attempted to fill multiple data and evidence gaps surrounding the debate. We determined a strong correlation between tolerance phenotypes and known genetic determinants of BC tolerance with an extensive set of fresh produce isolates. We assessed BC selection on L. monocytogenes through a large-scale and source-structured genomic survey of 25,083 publicly available L. monocytogenes genomes from diverse sources in the United States. With the consideration of processing environment constraints, we monitored the temporal onset and duration of adaptive BC tolerance in both tolerant and sensitive isolates. Finally, we examined residual BC concentrations throughout a fresh produce processing facility at different time points during daily operation. While genomic evidence supports elevated BC selection and the recommendation for sanitizer rotation in the general context of food processing environments, it also suggests a marked variation in the occurrence and potential impact of the selection among different commodities and sectors. For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes. IMPORTANCE Our study demonstrates an integrative approach to improve food safety assessment and control strategies in food processing environments through the collective leveraging of genomic surveys, laboratory assays, and processing facility sampling. In the example of assessing reduced Listeria susceptibility to a widely used sanitizer, this approach yielded multifaceted evidence that incorporates population genetic signals, experimental findings, and real-world constraints to help address a lasting debate of policy and practical importance.

Draft Genome Sequences of Multidrug-Resistant Escherichia coli Isolated from River Water.

The spread of antibiotic resistance poses a critical challenge worldwide. Contaminated environments can become reservoirs, spreading antibiotic-resistant bacteria and genetic determinants of resistance to humans directly or indirectly. Here, we report the draft genome sequence, the resistome, virulence genes, and sequence types of seven multidrug-resistant Escherichia coli strains isolated from river water.

Full-Length Transcriptomic Sequencing and Temporal Transcriptome Expression Profiling Analyses Offer Insights into Terpenoid Biosynthesis in Artemisia argyi.

Artemisiae argyi Folium is a traditional herbal medicine used for moxibustion heat therapy in China. The volatile oils in A.argyi leaves are closely related to its medicinal value. Records suggest that the levels of these terpenoids components within the leaves vary as a function of harvest time, with June being the optimal time for A. argyi harvesting, owing to the high levels of active ingredients during this month. However, the molecular mechanisms governing terpenoid biosynthesis and the time-dependent changes in this activity remain unclear. In this study, GC-MS analysis revealed that volatile oil levels varied across four different harvest months (April, May, June, and July) in A. argyi leaves, and the primarily terpenoids components (including both monoterpenes and sesquiterpenes) reached peak levels in early June. Through single-molecule real-time (SMRT) sequencing, corrected by Illumina RNA-sequencing (RNA-Seq), 44 full-length transcripts potentially involved in terpenoid biosynthesis were identified in this study. Differentially expressed genes (DEGs) exhibiting time-dependent expression patterns were divided into 12 coexpression clusters. Integrated chemical and transcriptomic analyses revealed distinct time-specific transcriptomic patterns associated with terpenoid biosynthesis. Subsequent hierarchical clustering and correlation analyses ultimately identified six transcripts that were closely linked to the production of these two types of terpenoid within A. argyi leaves, revealing that the structural diversity of terpenoid is related to the generation of the diverse terpene skeletons by prenyltransferase (TPS) family of enzymes. These findings can guide further studies of the molecular mechanisms underlying the quality of A. argyi leaves, aiding in the selection of optimal timing for harvests of A. argyi.

Subtyping Evaluation of Salmonella Enteritidis Using Single Nucleotide Polymorphism and Core Genome Multilocus Sequence Typing with Nanopore Reads.

Whole-genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing, such as Oxford nanopore technologies (ONT), presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of nanopore long read sequencing for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths in <7 h of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. IMPORTANCE Evaluation, standardization, and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high-quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. This study paves the way for evaluating and optimizing the logistics of implementing the ONT approach for foodborne pathogen surveillance in specific settings.