Genomic and Transcriptomic Analyses Reveal Multiple Strategies for Vibrio parahaemolyticus to Tolerate Sub-Lethal Concentrations of Three Antibiotics.

Vibrio parahaemolyticus can cause acute gastroenteritis, wound infections, and septicemia in humans. The overuse of antibiotics in aquaculture may lead to a high incidence of the multidrug-resistant (MDR) pathogen. Nevertheless, the genome evolution of V. parahaemolyticus in aquatic animals and the mechanism of its antibiotic tolerance remain to be further deciphered. Here, we investigated the molecular basis of the antibiotic tolerance of V. parahaemolyticus isolates (n = 3) originated from shellfish and crustaceans using comparative genomic and transcriptomic analyses. The genome sequences of the V. parahaemolyticus isolates were determined (5.0-5.3 Mb), and they contained 4709-5610 predicted protein-encoding genes, of which 823-1099 genes were of unknown functions. Comparative genomic analyses revealed a number of mobile genetic elements (MGEs, n = 69), antibiotic resistance-related genes (n = 7-9), and heavy metal tolerance-related genes (n = 2-4). The V. parahaemolyticus isolates were resistant to sub-lethal concentrations (sub-LCs) of ampicillin (AMP, 512 µg/mL), kanamycin (KAN, 64 µg/mL), and streptomycin (STR, 16 µg/mL) (p < 0.05). Comparative transcriptomic analyses revealed that there were significantly altered metabolic pathways elicited by the sub-LCs of the antibiotics (p < 0.05), suggesting the existence of multiple strategies for antibiotic tolerance in V. parahaemolyticus. The results of this study enriched the V. parahaemolyticus genome database and should be useful for controlling the MDR pathogen worldwide.

Visible-Light-Mediated Radical Hydroalkylative Cyclization of 1,6-Enynes.

A radical hydroalkylative cyclization approach accessing various alkenyl heterocyclic compounds was developed using dimethyl malonate and 1,6-enynes in the presence of visible-light photoredox catalysis. The use of Ir(dtbbpy)(ppy)2PF6 as a photosensitizer enables carbon atom radical formation and initiates the cascade cyclization reaction under mild conditions.

Modeling naturally-occurring Vibrio parahaemolyticus in post-harvest raw shrimps.

There is little known about the growth and survival of naturally-occurring Vibrio parahaemolyticus in harvested raw shrimps. In this study, the fate of naturally-occurring V. parahaemolyticus in post-harvest raw shrimps was investigated from 4℃ to 30℃ using real-time PCR combined with propidium monoazide (PMA-qPCR). The Baranyi-model was used to fit the growth and survival data. A square root model and non-linear Arrhenius model was then used to quantify the parameters derived from the Baranyi-model. The results showed that naturally-occurring V. parahaemolyticus were slowly inactivated at 4℃ and 7℃ with deactivation rates of 0.019 Log CFU/g/h and 0.025 Log CFU/g/h. Conversely, at 15, 20, 25, and 30 °C, the average maximum growth rates (µmax) of naturally-occurring V. parahaemolyticus were determined to be 0.044, 0.105, 0.179 and 0.336 Log CFU/g/h, accompanied by the average lag phases (λ) of 15.5 h, 7.3 h, 4.4 h and 3.7 h. The validation metrics, Af and Bf, for both the square root model and non-linear, indicating that the model had a good ability to predict the growth behavior of naturally-occurring V. parahaemolyticus in post-harvest raw shrimps. Furthermore, a comparative exploration between the growth of artificially contaminated V. parahaemolyticus in cooked shrimps and naturally-occurring V. parahaemolyticus in post-harvest raw shrimps revealed intriguing insights. While no substantial distinction in deactivation rates emerged at 4 °C and 7 °C (P > 0.05), a discernible disparity in growth rates was observable at 15 °C, 20 °C, 25 °C, and 30 °C, with the former surpassing the latter. Which indicated the risk of V. parahaemolyticus using models derived from cooked shrimps may be biased. Our study also unveiled a discernible seasonal effect. The µmax and λ of V. parahaemolyticus in shrimps harvested in summer were similar to those harvested in autumn, while the initial and maximum bacterial concentration harvested in summer were higher than those harvested in autumn. This predictive microbiology model of naturally-occurring V. parahaemolyticus in raw shrimps provides relevance to modelling growth in situ.

The two-component system expression patterns and immune regulatory mechanism of Vibrio parahaemolyticus with different genotypes at the early stage of infection in THP-1 cells.

Vibrio parahaemolyticus must endure various challenging circumstances while being swallowed by phagocytes of the innate immune system. Moreover, bacteria should recognize and react to environmental signals quickly in host cells. Two-component system (TCS) is an important way for bacteria to perceive external environmental signals and transmit them to the interior to trigger the associated regulatory mechanism. However, the regulatory function of V. parahaemolyticus TCS in innate immune cells is unclear. Here, the expression patterns of TCS in V. parahaemolyticus-infected THP-1 cell-derived macrophages at the early stage were studied for the first time. Based on protein-protein interaction network analysis, we mined and analyzed seven critical TCS genes with excellent research value in the V. parahaemolyticus regulating macrophages, as shown below. VP1503, VP1502, VPA0021, and VPA0182 could regulate the ATP-binding-cassette (ABC) transport system. VP1735, uvrY, and peuR might interact with thermostable hemolysin proteins, DNA cleavage-related proteins, and TonB-dependent siderophore enterobactin receptor, respectively, which may assist V. parahaemolyticus in infected macrophages. Subsequently, the potential immune escape pathways of V. parahaemolyticus regulating macrophages were explored by RNA-seq. The results showed that V. parahaemolyticus might infect macrophages by controlling apoptosis, actin cytoskeleton, and cytokines. In addition, we found that the TCS (peuS/R) could enhance the toxicity of V. parahaemolyticus to macrophages and might contribute to the activation of macrophage apoptosis. IMPORTANCE This study could offer crucial new insights into the pathogenicity of V. parahaemolyticus without tdh and trh genes. In addition, we also provided a novel direction of inquiry into the pathogenic mechanism of V. parahaemolyticus and suggested several TCS key genes that may assist V. parahaemolyticus in innate immune regulation and interaction.

Visible-Light Photoredox Catalyzed Regioselective 1,4-Hydroalkylation of 1,3-Enyne.

Described herein is a visible-light photoredox-catalyzed regioselective 1,4-hydroalkylation of 1,3-enynes. Various of di- and tri-substituent allenes were really accessible under the present reaction conditions. The visible-light photoredox activation of the carbon nucleophile to generate its radical species, allowing the addition with un-activated enynes. The synthetic utility for the present protocol was demonstrated by a large-scale reaction, as well as the derivatization of the allene product.

Discovery of Novel Resistance Mechanisms of Vibrio parahaemolyticus Biofilm against Aminoglycoside Antibiotics.

Inappropriate use of antibiotics eventually leads to the emergence of antibiotic-resistant strains and invalidates the treatment of infectious diseases. Aminoglycoside antibiotics (AGAs) are a class of broad-spectrum cationic antibiotics widely used for the treatment of Gram-negative bacterial infections. Understanding the AGA resistance mechanism of bacteria would increase the efficacy of treating these infections. This study demonstrates a significant correlation between AGA resistance and the adaptation of biofilms by Vibrio parahaemolyticus (VP). These adaptations were the result of challenges against the aminoglycosides (amikacin and gentamicin). Confocal laser scanning microscope (CLSM) analysis revealed an enclosure type mechanism where the biological volume (BV) and average thickness (AT) of V. parahaemolyticus biofilm were significantly positively correlated with amikacin resistance (BIC) (p < 0.01). A neutralization type mechanism was mediated by anionic extracellular polymeric substances (EPSs). The biofilm minimum inhibitory concentrations of amikacin and gentamicin were reduced from 32 µg/mL to 16 µg/mL and from 16 µg/mL to 4 µg/mL, respectively, after anionic EPS treatment with DNase I and proteinase K. Here, anionic EPSs bind cationic AGAs to develop antibiotic resistance. Transcriptomic sequencing revealed a regulatory type mechanism, where antibiotic resistance associated genes were significantly upregulated in biofilm producing V. parahaemolyticus when compared with planktonic cells. The three mechanistic strategies of developing resistance demonstrate that selective and judicious use of new antibiotics are needed to win the battle against infectious disease.

Transcriptome analysis of the biofilm formation mechanism of Vibrio parahaemolyticus under the sub-inhibitory concentrations of copper and carbenicillin.

Biofilm formation of Vibrio parahaemolyticus enhanced its tolerance to the environment, but caused many serious problems to food safety and human health. In this paper, the effects of copper and carbenicillin (CARB) stress on the formation of the biofilms of V. parahaemolyticus organisms were studied, and RNA sequencing technology was used to compare the differences in transcriptome profiles of the biofilm-related genes of V. parahaemolyticus organisms under different sub-inhibitory stresses. The results proved that V. parahaemolyticus had a large growth difference under the two stresses, copper and CARB at 1/2 minimal inhibitory concentration (MIC), and it could form a stable biofilm under both stress conditions. The amount of biofilm formed under CARB stress was significantly higher than that of copper stress (p < 0.05). Based on the analysis of transcriptome sequencing results 323, 1,550, and 1,296 significantly differential expressed genes were identified in the three treatment groups namely 1/2 MIC CARB, Cu2+, and Cu2++CARB. Through COG annotation, KEGG metabolic pathway analysis and gene expression analysis related to biofilm formation, the functional pathways of transcriptome changes affecting V. parahaemolyticus were different in the three treatment groups, and the CARB treatment group was significantly different from the other two groups. These differences indicated that the ABC transport system, two-component system and quorum sensing were all involved in the biofilm formation of the V. parahaemolytic by regulating flagellar motility, extracellular polysaccharides and extracellular polymer synthesis. Exploring the effects of different stress conditions on the transcriptome of V. parahaemolyticus could provide a basis for future research on the complex network system that regulates the formation of bacterial biofilms.

Antimicrobial resistomes in food chain microbiomes.

The safety and integrity of the global food system is in a constant state of flux with persistent chemical and microbial risks. While chemical risks are being managed systematically, microbial risks pose extra challenges. Antimicrobial resistant microorganism and persistence of related antibiotic resistance genes (ARGs) in the food chain adds an extra dimension to the management of microbial risks. Because the food chain microbiome is a key interface in the global health system, these microbes can affect health in many ways. In this review, we systematically summarize the distribution of ARGs in foods, describe the potential transmission pathway and transfer mechanism of ARGs from farm to fork, and discuss potential food safety problems and challenges. Modulating antimicrobial resistomes in the food chain facilitates a sustainable global food production system.

Expression patterns and influence of the two-component system in Vibrio parahaemolyticus of different genotypes.

Vibrio parahaemolyticus is a foodborne pathogen that threatens global food security and human health. The two-component system (TCS) is a primary method for bacteria self-regulate and adapt to the environment. Previous studies have shown that V. parahaemolyticus has four hemolytic genotypes with diverse biological phenotypes and environmental adaptability, but the mechanism is unclear. In this study, we investigated TCS expression patterns in V. parahaemolyticus with different genotypes for the first time and explored the differences in TCS between strains. The results showed similarities in the TCS expression pattern between VPC17 (tdh+/trh-) and VPC44 (tdh-/trh-), while VPC85(tdh-/trh+) had the least similar TCS expression pattern to the other three strains. Analysis of biological information revealed that different regulations of C4 dicarboxylate transport, tetrathionate uptake, antibiotic resistance, and flagellar synthesis involved in the TCS might influence strains' growth, antibiotic resistance, biofilm, and virulence. The different TCS regulatory abilities of strains might be one of the reasons for diverse biological characteristics and different environmental adaptations. This work provides a theoretical basis and a new research direction for the strain variability of V. parahaemolyticus.

Hierarchical Flower-like WO3 Nanospheres Decorated with Bimetallic Au and Pd for Highly Sensitive and Selective Detection of 3-Hydroxy-2-butanone Biomarker.

Listeria monocytogenes, which is abundant in environment, can lead to many kinds of serious illnesses and even death. Nowadays, indirectly detecting the metabolite biomarker of L. monocytogenes, 3-hydroxy-2-butanone, has been verified to be an effective way to evaluate the contamination of L. monocytogenes. However, this detection approach is still limited by sensitivity, selectivity, and ppb-level detection limit. Herein, low-cost and highly sensitive and selective 3-hydroxy-2-butanone sensors have been proposed based on the bimetallic AuPd decorated hierarchical flower-like WO3 nanospheres. Notably, the 1.0 wt % AuPd-WO3 based sensors displayed the highest sensitivity (Ra/Rg = 84 @ 1 ppm) at 250 °C. In addition, the sensors showed outstanding selectivity, rapid response/recovery (8/4 s @ 10 ppm), and low detection limit (100 ppb). Furthermore, the evaluation of L. monocytogenes with high sensitivity and specificity has been achieved using 1.0 wt % AuPd-WO3 based sensors. Such a marvelous sensing performance benefits from the synergistic effect of bimetallic AuPd nanoparticles, which lead to thicker electron depletion layer and increased adsorbed oxygen species. Meanwhile, the unique hierarchical nanostructure of the flower-like WO3 nanospheres benefits the gas-sensing performance. The AuPd-WO3 nanosphere-based sensors exhibit a particular and highly selective method to detect 3-hydroxy-2-butanone, foreseeing a feasible route for the rapid and nondestructive evaluation of foodborne pathogens.

Copper-Catalyzed Oxidative Dearomatized Oxyalkylation of Indoles with Alcohols: Synthesis of 3-Alkoxy-2-Oxindoles.

Described herein is a copper-catalyzed efficient oxidative dearomatized functionalization of indoles by using alcohols as the nucleophiles. Various 3-alkoxy-2-oxindoles were accessible with good isolated yields. The synthetic potential applications are demonstrated by the large-scale reaction, as well as the derivatization of the desired 3-alkoxy substituted-2-oxindole products.

Current advances of Pichia pastoris as cell factories for production of recombinant proteins.

Pichia pastoris (syn. Komagataella spp.) has attracted extensive attention as an efficient platform for recombinant protein (RP) production. For obtaining a higher protein titer, many researchers have put lots of effort into different areas and made some progress. Here, we summarized the most recent advances of the last 5 years to get a better understanding of its future direction of development. The appearance of innovative genetic tools and methodologies like the CRISPR/Cas9 gene-editing system eases the manipulation of gene expression systems and greatly improves the efficiency of exploring gene functions. The integration of novel pathways in microorganisms has raised more ideas of metabolic engineering for enhancing RP production. In addition, some new opportunities for the manufacture of proteins have been created by the application of novel mathematical models coupled with high-throughput screening to have a better overview of bottlenecks in the biosynthetic process.

Gait Recognition by Combining the Long-Short-Term Attention Network and Personal Physiological Features.

Although gait recognition has been greatly improved by efforts from many researchers in recent years, its performance is still unsatisfactory due to the lack of gait information under the real scenariowhere only one or two images may be used for recognition. In this paper, a new gait recognition framework is brought about which can combine the long-short-term attention modules on silhouette images over the whole sequence and the real human physiological information calculated by a monocular image. The contributions of this work include the following: (1) Fusing the global long-term attention (GLTA) and local short-term attention (LSTA) over the whole query sequence to improve the gait recognition accuracy, where both the short-term gait feature (from two or three frames) and long-term feature (from the whole sequence) are extracted; (2) presenting a method to calculate the real personal static and dynamic physiological features through a single monocular image; (3) by efficiently applying the human physiological information, a new physiological feature extraction (PFE) network is proposed to concatenate the physiological information with silhouette for gait recognition. Through the experiments between the CASIA-B and Multi-state Gait datasets, the effectiveness and efficiency of the proposed method are proven. Under three different walking conditions of the CASIA-B dataset, the mean accuracy of rank-1 in our method is up to 89.6%, and in the Multi-state Gait dataset, wearing different clothes, the mean accuracy of rank-1 in our method is 2.4% higher than the other works.

Natural Inhibitors Targeting the Localization of Lipoprotein System in Vibrio parahaemolyticus.

The localization of lipoprotein (Lol) system is responsible for the transport of lipoproteins in the outer membrane (OM) of Vibrio parahaemolyticus. LolB catalyzes the last step in the Lol system, where lipoproteins are inserted into the OM. If the function of LolB is impeded, growth of V. parahaemolyticus is inhibited, due to lack of an intact OM barrier for protection against the external environment. Additionally, it becomes progressively harder to generate antimicrobial resistance (AMR). In this study, LolB was employed as the receptor for a high-throughput virtual screening from a natural compounds database. Compounds with higher glide score were selected for an inhibition assay against V. parahaemolyticus. It was found that procyanidin, stevioside, troxerutin and rutin had both exciting binding affinity with LolB in the micromolar range and preferable antibacterial activity in a concentration-dependent manner. The inhibition rates of 100 ppm were 87.89%, 86.2%, 91.39% and 83.71%, respectively. The bacteriostatic mechanisms of the four active compounds were explored further via fluorescence spectroscopy and molecular docking, illustrating that each molecule formed a stable complex with LolB via hydrogen bonds and pi-pi stacking interactions. Additionally, the critical sites for interaction with V. parahaemolyticus LolB, Tyr108 and Gln68, were also illustrated. This paper demonstrates the inhibition of LolB, thus, leading to antibacterial activity, and identifies LolB as a promising drug target for the first time. These compounds could be the basis for potential antibacterial agents against V. parahaemolyticus.

Antibacterial activity of essential oils extracted from the unique Chinese spices cassia bark, bay fruits and cloves.

Spices are widely used in daily life such as diet and have certain activity. Especially in China, spices have been mainly used as condiments for thousands of years in order to improve the sensory quality of food; in addition, they and their derivatives can also be used as preservatives. In this study, three spices with unique Chinese characteristics widely used were selected: cassia bark (bark of Cinnamomum camphora Presl), bay fruits (Laurus nobilis), and cloves (Syzygiumaromaticum). The main components and antibacterial ability of these three spices were analyzed by simulated extraction method. Through headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrometry (GC-MS) analysis, it was determined that the main active compounds in the essential oils of cassia bark, bay fruits and cloves were cinnamaldehyde (78.11%), cinnamaldehyde (61.78%) and eugenol (75.23%), respectively. The agar plate diffusion test and the simulated food culture medium experiment confirmed that the essential oils extracted from the three flavors have antibacterial effects on Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Listeria ivanovii, Listeria grayi and Vibrio parahaemolyticus. The antibacterial activity of different strains has different optimal extraction conditions. Generally speaking, cinnamon essential oil has the strongest antibacterial activity, while laurel fruit has the lowest antibacterial activity. The study proved the antibacterial activity of these three Chinese-specific spices and provided some new ideas and methods for the subsequent research and preparation of natural food additives and food antibacterial agents.

[Cascaded multi-level medical image registration method based on transformer].

In deep learning-based image registration, the deformable region with complex anatomical structures is an important factor affecting the accuracy of network registration. However, it is difficult for existing methods to pay attention to complex anatomical regions of images. At the same time, the receptive field of the convolutional neural network is limited by the size of its convolution kernel, and it is difficult to learn the relationship between the voxels with far spatial location, making it difficult to deal with the large region deformation problem. Aiming at the above two problems, this paper proposes a cascaded multi-level registration network model based on transformer, and equipped it with a difficult deformable region perceptron based on mean square error. The difficult deformation perceptron uses sliding window and floating window techniques to retrieve the registered images, obtain the difficult deformation coefficient of each voxel, and identify the regions with the worst registration effect. In this study, the cascaded multi-level registration network model adopts the difficult deformation perceptron for hierarchical connection, and the self-attention mechanism is used to extract global features in the basic registration network to optimize the registration results of different scales. The experimental results show that the method proposed in this paper can perform progressive registration of complex deformation regions, thereby optimizing the registration results of brain medical images, which has a good auxiliary effect on the clinical diagnosis of doctors.

[Animal models in bacterial biofilm research: a review].

Biofilm formation is closely related to pathogenicity and antibiotic resistance of bacteria, and plays important roles in a number of chronic and subchronic infections. Animal models are widely used in the research of bacterial biofilm-associated infections, and provide a powerful scientific tool for investigating its pathogenesis and control strategies. This review summarized the application of mammalian models (e.g. mouse, rabbit, and pig) and non-mammalian models (e.g. Drosophila melanogaster, Zebrafish, and Caenorhabditis elegans) in bacterial biofilm studies, and prospects the application of animal models in biofilm. This review may facilitate the selection of suitable animal models in the study of biofilm-associated infections, so as to prevent and control the potential adverse effects.

Meta-Analysis for the Global Prevalence of Foodborne Pathogens Exhibiting Antibiotic Resistance and Biofilm Formation.

Antimicrobial-resistant (AMR) foodborne bacteria causing bacterial infections pose a serious threat to human health. In addition, the ability of some of these bacteria to form biofilms increases the threat level as treatment options may become compromised. The extent of antibiotic resistance and biofilm formation among foodborne pathogens remain uncertain globally due to the lack of systematic reviews. We performed a meta-analysis on the global prevalence of foodborne pathogens exhibiting antibiotic resistance and biofilm formation using the methodology of a Cochrane review by accessing data from the China National Knowledge Infrastructure (CNKI), PubMed, and Web of Science databases between 2010 and 2020. A random effects model of dichotomous variables consisting of antibiotic class, sample source, and foodborne pathogens was completed using data from 332 studies in 36 countries. The results indicated AMR foodborne pathogens has become a worrisome global issue. The prevalence of AMR foodborne pathogens in food samples was greater than 10% and these foodborne pathogens were most resistant to ß-lactamase antibiotics with Bacillus cereus being most resistant (94%). The prevalence of AMR foodborne pathogens in human clinical specimens was greater than 19%, and the resistance of these pathogens to the antibiotic class used in this research was high. Independently, the overall biofilm formation rate of foodborne pathogenic bacteria was 90% (95% CI, 68%-96%) and a direct linear relationship between biofilm formation ability and antibiotic resistance was not established. Future investigations should document both AMR and biofilm formation of the foodborne pathogen isolated in samples. The additional information could lead to alternative strategies to reduce the burden cause by AMR foodborne pathogens.

Determination of arsenic in Chinese mitten crabs (Eriocheir sinensis): Effects of cooking and gastrointestinal digestion on food safety.

This study used the sexually mature Chinese mitten crabs as the research object, and the total arsenic and six major species were determined by high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The influence of three common cooking methods on the forms of arsenic and content in different edible parts of crabs were explored. Furthermore, the bioavailability of arsenic and its different forms in the crabs were studied by in vitro gastrointestinal digestion. Based on the risk assessment of arsenic in gastrointestinal digestion, the results showed that the Target Hazard Quotient (THQ) of arsenic was less than 1, which did not pose a significant health risk, but the risk of carcinogenesis of arsenic can not be ignored. Considering the effects of cooking and gastrointestinal digestion into account, a more realistic estimate of the risk associated with the consumption of Chinese mitten crabs can be deciphered.

Ultra-efficient trimethylamine gas sensor based on Au nanoparticles sensitized WO3 nanosheets for rapid assessment of seafood freshness.

Trimethylamine (TMA) is an organic amine gas used as an important index to assess the seafood freshness. In this work, an ultra-efficient trimethylamine gas sensor was constructed by ultrasonic treatment loading of Au nanoparticles (∼4 nm) on WO3 nanosheets prepared by solvothermal self-assembly method. The response of the sensor to 25 ppm TMA was as high as 217.72 (Rair/Rgas) at 300 ℃. In addition, the Au/WO3 nanosheets sensor exhibited rapid response-recovery time (8 s/6 s), low detection limit (0.5 ppm), and high selective detection of TMA. Moreover, the composition of volatiles produced by decay of Larimichthys crocea (0-15 days) was examined, which proved that the detection of TMA by the Au/WO3 sensor can evaluate the freshness of Larimichthys crocea. Such a magnificent gas sensing performance reveals that the Au/WO3 sensor has remarkable application potential in rapid and non-destructive seafood freshness assessment on the spot.