Optimization of hydrolysis conditions of alginate based on high performance liquid chromatography.

Alginate is the most abundant polysaccharide compound in brown algae, which is widely used in various fields. At present, the determination of the content of alginate is mostly carried out using sulfuric acid and trifluoroacetic acid hydrolysis followed by the determination of the content, but the results are not satisfactory, and there are problems such as low hydrolysis degree and low recovery rate. Therefore, in this study, based on the optimization of high performance liquid chromatographic conditions for pre-column derivatization of 1-phenyl-3-methyl-5-pyrazolone (PMP), the hydrolysis effects of sulfuric acid, trifluoroacetic acid (TFA), oxalic acid, and formic acid were compared and the hydrolysis conditions were optimized. The results showed that formic acid was the best hydrolyzing acid. The optimal hydrolysis conditions were 95 % formic acid at 110 °C for 10 h. The hydrolysis effect was stable, with high recovery and low destruction of monosaccharides, which made it possible to introduce formic acid into the subsequent polysaccharide hydrolysis. The pre-column derivatization high performance liquid chromatography method established in this study was accurate and reliable, and the hydrolysis acid with better effect was screened, which provided a theoretical basis for the subsequent determination of alginate content.

Characterization of the intestinal transport mechanism of polystyrene microplastics (MPs) and the potential inhibitory effect of green tea extracts on MPs intestinal absorption.

The aims of the current study included characterizing the intestinal transport mechanism of polystyrene microplastics (MPs) with different charges and sizes in the intestinal epithelial cell model and determining the inhibitory effect of green tea extracts (GTEs) on the intestinal absorption of MPs in Caco-2 cells. The smaller sizes, which included diameters of 0.2 µm, of amine-modified MPs compared to either larger size (1 µm diameter, or carboxylate-MPs (0.2 and 1 µm diameter) significantly lowered the cell viability of caco-2 cells that were measured by MTT assay (p < 0.05). The transported amount (particles/mL of the cell media) of amine-modified MPs by the Caco-2 cell, was not dependent according to the concentrations, energy, or temperature, but it was higher than the carboxylate-modified MPs. The co-treatment of GTEs with the amine-modified MPs inhibited Caco-2 cell cytotoxicity as well as reduced the production of intracellular reactive oxygen species (ROS) in HepG2 generated by the exposure of amine-modified MPs. The GTEs co-treatment also increased trans-epithelial electrical resistances (TEER) and reduced the transportation of Lucifer Yellow via the Caco-2 monolayer compared to only the amine-modified MPs exposure. The GTEs treatment led to a decrease in the number of amine-modified MPs transported to the basal side of the Caco-2 monolayer. The results from our study suggest that the consumption of GTEs could enhance the intestinal barrier function by recovering intestinal epithelial cell damage induced by MPs, which resulted in a decrease of the intestinal absorption of MPs.

Effect of increased pCO2 and temperature on the phytoplankton community in the coastal of Yellow Sea.

In order to study the dynamics of marine phytoplankton communities in response to anticipated in temperature and CO2, a shipboard continuous culture experiment (Ecostat) was conducted. The experiment involved simulations under current atmospheric CO2 concentrations (400 ppm) and projected year-2100 CO2 levels (1000 ppm), as well as varying temperature under present (22 °C) versus increased temperature (26 °C) in the Yellow Sea during the summer of 2020. The results showed that both the increased pCO2 and temperature had significant effects on microphytoplankton and picophytoplankton, with the warming effect proving to be more significant. The different responses of various species to acidification and warming and their coupling effect led to the changes in microphytoplankton and picophytoplankton community structure. Elevated temperature and greenhouse treatments promoted the growth of dominant diatoms and Synechococcus, such as Guinardia flaccida and Pseudo-nitzschia delicatissima. This phenomenons widened the ecological niche, and the changes in the growth patterns of dominant species consequently influenced the content of cellular elements. Mantel's analysis further demonstrated that both warming and greenhouse promoted the growth of diatoms and Synechococcus. Projections of marine phytoplankton community trends by the end of the century based on Growth Rate Ratio (GRR), indicated that not only would species with GRR < 1 decrease, but also numerous species with growth rates >1 at elevated pCO2 levels would be ousted from competition. This experiment demonstrates the need to investigate whether extended exposure to increased pCO2 and temperature over more extended time scales would similarly induce shifts in the biological and biogeochemical dynamics of the Yellow Sea.

Impact of environmental variables on the distribution of phytoplankton communities in the Southern Yellow Sea.

To gain a comprehensive understanding of the seasonal variation in the structure of phytoplankton communities in the Southern Yellow Sea (SYS), two research expeditions were conducted from 12 to 24 in April 2019, and from 12 to 22 in October of 2019. During the spring season, the phytoplankton community within the SYS was primarily comprised of diatoms and dinoflagellates, while in autumn, diatoms and cyanobacteria dominated. Thalassiosira rotula and Paralia sulcata were the dominant species in both seasons. In spring, P. sulcata displayed no obvious correlation with any environmental parameter, while in autumn, it exhibited negative correlations with environmental factors. According to the cluster and multidimensional scaling analyses, the phytoplankton community was stratified into three distinct ecological provinces in the SYS: the Western Yellow Sea, the Yellow Sea basin, and the southern coastal region. The phytoplankton community composition was predominantly affected by seasonal fluctuations in temperature and nutrient levels. Notably, the Yellow Sea basin exhibited the lowest phytoplankton abundance, largely because of the impact of the Yellow Sea Cold Water Mass. Furthermore, the presence of cyanobacteria, particularly prevalent in the Yellow Sea basin, may have been facilitated by transport mechanisms associated with the Kuroshio current. Aggregated boosted tree (ABT) and Generalized Additive models (GAM) suggested that temperature, DIN, salinity, and DIP were significant parameters of phytoplankton abundance in SYS. Additionally, the N:P nutrient ratio was a key parameter in governing the structure of phytoplankton communities during both seasons.

Temperature driving vertical stratification regulates phytoplankton community structure in the Bohai Sea and Yellow Sea.

To analyse the effects of physicochemical factors on the phytoplankton community in the Bohai Sea (BS) and Yellow Sea (YS), a investigation was conducted during 27 July to 10 August 2020. A sum of 156 species were identified in the BS and YS, including Bacillariophyta (69 species), Pyrrophyta (85 species) and Chrysophyta (2 species). The phytoplankton community were divided into four provinces according to Bray-Curtis similarity. In order to study the phytoplankton community in the BS and YS, we studied the phytoplankton community composition and their assembly mechanisms. The results showed that stochastic ecological processes had a greater effect on the province C community structure. The Raup-crick dissimilarity showed that deterministic factors had a greater effect on the province A, B and D communities structure. The habitat niche width results indicated that niche was larger in the province D, compared to the province A, B and C. Based on a structural equation model (SEM), we analyzed the effects of physicochemical factors on phytoplankton community structure and temperature was found to affect the phytoplankton community composition and structure by the vertical stratification. The result showed that temperature was an important parameter for phytoplankton abundance and revealed that temperature affected phytoplankton community structure by influencing the vertical stratification index (VSI) in the BS and YS.

Sulfated Galactans from Agarophytes: Review of Extraction Methods, Structural Features, and Biological Activities.

Agarophytes are important seaweeds of the Rhodophyta type, which have been highly exploited for industrial use as sources of a widely consumed polysaccharide of agar. In addition to that, sulfated galactans (SGs) from agarophytes, which consist of various functional sulfate groups, have attracted the attention of scientists in current studies. SGs possess various biological activities, such as anti-tumor, anticoagulant, anti-inflammatory, antioxidant, anti-obesity, anti-diabetic, anti-microbial, anti-diarrhea, and gut microbiota regulation properties. Meanwhile, the taxonomy, ecological factors, i.e., environmental factors, and harvest period, as well as preparation methods, i.e., the pretreatment, extraction, and purification conditions, have been found to influence the chemical compositions and fine structures of SGs, which have, further, been shown to have an impact on their biological activities. However, the gaps in the knowledge of the properties of SGs due to the above complex factors have hindered their industrial application. The aim of this paper is to collect and systematically review the scientific evidence about SGs and, thus, to pave the way for broader and otherwise valuable industrial applications of agarophytes for human enterprise. In the future, this harvested biomass could be sustainably used not only as a source of agar production but also as natural materials in functional food and pharmaceutical industries.

Rapid Mycobacterium abscessus antimicrobial susceptibility testing based on antibiotic treatment response mapping via Raman Microspectroscopy.

OBJECTIVES: Antimicrobial susceptibility tests (ASTs) are pivotal tools for detecting and combating infections caused by multidrug-resistant rapidly growing mycobacteria (RGM) but are time-consuming and labor-intensive. DESIGN: We used a Mycobacterium abscessus-based RGM model to develop a rapid (24-h) AST from the beginning of the strain culture, the Clinical Antimicrobials Susceptibility Test Ramanometry for RGM (CAST-R-RGM). The ASTs obtained for 21 clarithromycin (CLA)-treated and 18 linezolid (LZD)-treated RGM isolates. RESULTS: CAST-R-RGM employs D2O-probed Raman microspectroscopy to monitor RGM metabolic activity, while also revealing bacterial antimicrobial drug resistance mechanisms. The results of clarithromycin (CLA)-treated and linezolid (LZD)-treated RGM isolates exhibited 90% and 83% categorical agreement, respectively, with conventional AST results of the same isolates. Furthermore, comparisons of time- and concentration-dependent Raman results between CLA- and LZD-treated RGM strains revealed distinct metabolic profiles after 48-h and 72-h drug treatments, despite similar profiles obtained for both drugs after 24-h treatments. CONCLUSIONS: Ultimately, the rapid, accurate, and low-cost CAST-R-RGM assay offers advantages over conventional culture-based ASTs that warrant its use as a tool for improving patient treatment outcomes and revealing bacterial drug resistance mechanisms.

Bioactivities of the Popular Edible Brown Seaweed Sargassum fusiforme: A Review.

Sargassum fusiforme has a wide range of active constituents (such as polysaccharides, sterols, polyphenols, terpenes, amino acids, trace elements, etc.) and is an economically important brown algae with a long history. In recent years, S. fusiforme has been intensively studied and has attracted wide attention in the fields of agriculture, environment, medicine, and functional food. In this review, we reviewed the current research status of S. fusiforme at home and abroad over the past decade by searching Web of science, Google Scholar, and other databases, and structurally analyzed the active components of S. fusiforme, and on this basis, we focused on summarizing the cutting-edge research and scientific issues on the role of various active substances in S. fusiforme in exerting antioxidant, anti-inflammatory, antitumor, antidiabetic, immunomodulatory, antiviral antibacterial, and anticoagulant effects. The mechanisms by which different substances exert active effects were further summarized by exploring different experimental models and are shown visually. It provides a reference to promote further development and comprehensive utilization of S. fusiforme resources.

IS26 mediated blaCTX-M-65 amplification in Escherichia coli increase the antibiotic resistance to cephalosporin in vivo.

OBJECTIVES: To characterize two Escherichia coli strains isolated from a patient pre- and post-treatment, using ß-lactams and ß-lactam/ß-lactamase inhibitor combinations (BLBLIs). METHODS: A combination of antibiotic susceptibility testing (AST) with whole genome sequencing using Illumina and Oxford Nanopore platforms. Long-read sequencing and reverse transcription-quantitative PCR were performed to determine the copy numbers and expression levels of antibiotic resistance genes (ARGs), respectively. Effect on fitness costs were assessed by growth rate determination. RESULTS: The strain obtained from the patient after the antibiotic treatment (XH989) exhibited higher resistance to cefepime, BLBLIs and quinolones compared with the pre-treatment strain (XH987). Sequencing revealed IS26-mediated duplications of a IS26-fosA3-blaCTX-M-65 plasmid-embedded element in strain XH989. Long-read sequencing (7.4 G data volume) indicated a variation in copy numbers of blaCTX-M-65 within one single culture of strain XH989. Increased copy numbers of the IS26-fosA3-blaCTX-M-65 element were correlated with higher CTX-M-65 expression level and did not impose fitness costs, while facilitating faster growth under high antibiotic concentrations. CONCLUSION: Our study is an example from the clinic how BLBLIs and ß-lactams exposure in vivo possibly promoted the amplification of an IS26-multiple drug resistance (MDR) region. The observation of a copy number variation seen with the blaCTX-M-65 gene in the plasmid of the post-treatment strain expands our knowledge of insertion sequence dynamics and evolution during treatment.

Efficacy of denosumab on bone metabolism and bone mineral density in renal transplant recipients: A systematic review and meta-analysis.

BACKGROUND: Post-transplant bone disease (PTBD) is a common complication in kidney transplant recipients. This systematic review and meta-analysis evaluates the efficiency and safety of denosumab for the treatment of PTBD in kidney transplant recipients. METHODS: Comprehensive search of PubMed Central, SCOPUS, EMBASE, MEDLINE, Cochrane trial registry, Google Scholar, and Clinicaltrials.gov databases was done for studies, published until April 2023. Primary outcomes included changes in bone mineral density (BMD) and T-scores. Secondary outcomes included incidence of fractures, alterations in bone turnover markers, and the incidence of adverse events. RESULTS: Eleven studies with a total of 511 participants that underwent kidney transplant were included. Denosumab treatment resulted in a significant improvement in lumbar spine BMD (SMD: -0.31, 95% CI: -0.56 to -0.06) and T-score (SMD: -1.07, 95% CI: -1.51 to -0.64), while no differences were detected in hip/femoral neck BMD and the T-score. There was no marked change in the fracture incidence (OR: 0.42, 95% CI: 0.06 to 3.07). However, patients who received denosumab treatment had an increased incidence rate of hypocalcemia (OR: 9.98, 95% CI: 1.72 to 57.88). CONCLUSIONS: Denosumab treatment may improve lumbar spine BMD and T-scores in patients with PTBD. However, it does not significantly affect fracture incidence and may increase the risk of hypocalcemia. These findings underline the necessity for well-powered, randomized controlled trials to further clarify the role of denosumab in managing PTBD.

Anti-Melanogenesis and Anti-Photoaging Effects of the Sulfated Polysaccharides Isolated from the Brown Seaweed Padina boryana.

Sulfated polysaccharides isolated from seaweeds are thought of as ideal ingredients in the pharmaceutical, nutraceutical, and cosmetics industries. Our previous study isolated and characterized sulfated polysaccharides from Padina boryana. The sulfated polysaccharides of Padina boryana (PBP) were extracted, and the antioxidant activity of PBP was evaluated. The results indicate that PBP possesses antioxidant effects and potential in the cosmetic industry. To further investigate the potential of PBP in cosmetics, the photoprotective and anti-melanogenesis effects of PBP were evaluated. The anti-melanogenesis test results display that PBP reduced the melanin content in the murine melanoma cells stimulated by alpha melanocyte-stimulating hormone from 203.7% to 183.64%, 144.63%, and 127.57% at concentrations of 25 µg/mL, 50 µg/mL, and 100 µg/mL, respectively. The anti-photodamage test results showed that PBP significantly protected skin cells against UVB-stimulated photodamage. PBP suppressed human epidermal keratinocyte (HaCaT cell) death by inhibiting apoptosis and reducing the level of intracellular reactive oxygen species. The intracellular reactive oxygen species level of HaCaT cells irradiated by UVB was reduced from 192.67% to 181.22%, 170.25%, and 160.48% by 25 µg/mL, 50 µg/mL, and 100 µg/mL PBP, respectively. In addition, PBP remarkably reduced UVB-induced human dermal fibroblast damage by suppressing oxidative damage, inhibiting collagen degradation, and attenuating inflammatory responses. These results indicate that PBP possesses photoprotective and anti-melanogenesis activities and suggest that PBP is a potential ingredient in the cosmetic industry.

In Vitro and In Vivo Protective Effects of Agaro-Oligosaccharides against Hydrogen Peroxide-Stimulated Oxidative Stress.

In our previous research, we investigated the anti-inflammatory activity of the agaro-oligosaccharides prepared from the agar of Gracilaria lemaneiformis (AO). In the present study, in order to further explore the bioactivities of AO, the antioxidant activity of AO was investigated in vitro in Vero cells and in vivo in zebrafish. AO scavenged alkyl, 1,1-diphenyl-2-picrylhydrazyl, and hydroxyl radicals at the IC50 value of 4.86 ± 0.13, 3.02 ± 0.44, and 1.33 ± 0.05 mg/mL, respectively. AO significantly suppressed hydrogen peroxide (H2O2)-stimulated oxidative damage by improving cell viability. This happened via suppressing apoptosis by scavenging intracellular reactive oxygen species (ROS). Furthermore, the in vivo results displayed that AO protected zebrafish against H2O2-stimulated oxidative damage by reducing the levels of intracellular ROS, cell death, and lipid peroxidation in a dose-dependent manner. These results indicate that AO effectively alleviated in vitro and in vivo oxidative damage stimulated by H2O2, and suggest the potential of AO in the cosmetic and functional food industries.

Protective Effect of Sargassum fusiforme Fucoidan against Ethanol-Induced Oxidative Damage in In Vitro and In Vivo Models.

Our previous studies have evaluated the bioactivities of a fucoidan isolated from Sargassum fusiforme (SF-F). To further investigate the health benefit of SF-F, in the present study, the protective effect of SF-F against ethanol (EtOH)-induced oxidative damage has been evaluated in in vitro and in vivo models. SF-F effectively improved the viability of EtOH-treated Chang liver cells by suppressing apoptosis. In addition, the in vivo test results indicate that SF-F significantly and dose-dependently increased the survival rate of zebrafish treated with EtOH. Further research results show that this action works through decreasing cell death via reduced lipid peroxidation by scavenging intracellular reactive oxygen species in EtOH-stimulated zebrafish. These results indicate that SF-F effectively protected Chang liver cells and zebrafish against EtOH-induced oxidative damage and suggest the potential of SF-F to be used as an ingredient in the functional food industry.

Stellar initial mass function varies with metallicity and time.

Most structural and evolutionary properties of galaxies strongly rely on the stellar initial mass function (IMF), namely the distribution of the stellar mass formed in each episode of star formation1-4. The IMF shapes the stellar population in all stellar systems, and so has become one of the most fundamental concepts of modern astronomy. Both constant and variable IMFs across different environments have been claimed despite a large number of theoretical5-7 and observational efforts8-15. However, the measurement of the IMF in Galactic stellar populations has been limited by the relatively small number of photometrically observed stars, leading to high uncertainties12-16. Here we report a star-counting result based on approximately 93,000 spectroscopically observed M-dwarf stars, an order of magnitude more than previous studies, in the 100-300 parsec solar neighbourhood. We find unambiguous evidence of a variable IMF that depends on both metallicity and stellar age. Specifically, the stellar population formed at early times contains fewer low-mass stars compared with the canonical IMF, independent of stellar metallicities. In more recent times, however, the proportion of low-mass stars increases with stellar metallicity. The variable abundance of low-mass stars in our Milky Way establishes a powerful benchmark for models of star formation and can heavily affect results in Galactic chemical-enrichment modelling, mass estimation of galaxies and planet-formation efficiency.

Antioxidant and anti-photoaging effects of a fucoidan isolated from Turbinaria ornata.

Fucoidans isolated from brown seaweeds are potential ingredients in the cosmetic industry. In our preosvious study, a fucoidan was isolated from the brown seaweed Turbinaria ornata (TO-F10) and the anti-inflammatory effect of TO-F10 was evaluated. In order to further explore the potential of TO-F10 in cosmetics, in the present study, antioxidant and photoprotective effects of TO-F10 were investigated. TO-F10 remarkably protected Vero cells against AAPH-stimulated cell death by reducing apoptosis via scavenging intracellular reactive oxygen species (ROS). In addition, TO-F10 increased the survival rate of AAPH-treated zebrafish by suppressing oxidative stress displayed in reducing the levels of ROS, cell death, and lipid peroxidation. Furthermore, TO-F10 effectively attenuated UVB-induced in vitro and in vivo photodamage. TO-F10 increased the viability of UVB-irradiated human keratinocytes via suppressing apoptosis by reducing the intracellular ROS level. Besides, TO-F10 effectively attenuated in vivo photodamage stimulated by UVB irradiation via inhibiting oxidative stress and inflammatory response in zebrafish. These results demonstrate that TO-F10 possesses in vitro and in vivo antioxidant and photoprotective effects, and suggest TO-F10 is a potential ingredient in the cosmetic industry.

Impact of Monascus purpureus fermentation on antioxidant activity, free amino acid profiles and flavor properties of kelp (Saccharina japonica).

This study evaluated the efficacy of Monascus purpureus fermentation on Saccharina japonica (SJ). Healthy substances and antioxidant activity of fermented SJ (FSJ) were determined. Results showed that fermentation caused the release of phenolic compounds and flavonoids, which resulted in the enhancement of antioxidant activity. Essential amino acids and γ-aminobutyric acid also greatly accumulated in FSJ. Sensory evaluation and gas chromatography-ion mobility spectrometry (GC-IMS) were used to evaluate flavor properties of FSJ. A lexicon consisted of 24 descriptors was established for SJ and FSJ, of which 14 descriptors were regarded as odor attributes. A total of 46 volatile compounds were identified by GC-IMS and showed positive correlation with odor attributes. Fifteen volatile compounds were screened as key compounds, tricarboxylic acid cycle, embden-meyerhof-parnas and amino acid catabolism were main formation metabolisms of them. Advanced properties of FSJ indicated that fermentation is a promising approach for the production of SJ food.

Anti-inflammatory effect of fucoidan isolated from fermented Sargassum fusiforme in in vitro and in vivo models.

Fucoidans possess potent anti-inflammatory effects. In the present study, the anti-inflammatory effect of the fucoidan (SFF-PS-F5) isolated from fermented Sargassum fusiforme was evaluated in vitro in RAW 264.7 macrophages and in vivo in zebrafish. The in vitro test results demonstrate that SFF-PS-F5 effectively inhibited nitric oxide (NO) production induced by lipopolysaccharides (LPS) in RAW 264.7 cells. SFF-PS-F5 effectively and concentration-dependently improved the viability of LPS-stimulated RAW 264.7 cells, and reduced the level of prostaglandin E2, interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6. Further results display that these effects were actioned by suppressing the expression of inducible nitric oxide synthase and cyclooxygenase-2 via regulating the nuclear factor kappa-B signaling pathway. The in vivo test results indicate that SFF-PS-F5 remarkably reduced reactive oxygen species, cell death, and NO levels in LPS-treated zebrafish. These results indicate that SFF-PS-F5 could inhibit both in vitro and in vivo inflammatory responses and suggest it is a functional ingredient in the functional food and cosmetic industries.

Anti-Melanogenesis and Photoprotective Effects of Ecklonia maxima Extract Containing Dieckol and Eckmaxol.

Seaweeds are potential ingredients in the cosmeceutical industry. Our previous study demonstrates that the phlorotannin-enriched extract of Ecklonia maxima (EME-EA) containing dieckol and eckmaxol possesses strong anti-inflammatory activity and suggests the cosmeceutical potential of EME-EA. In order to evaluate the cosmeceutical potential of EME-EA, the anti-melanogenesis and photoprotective effects of EME-EA were investigated in this study. EME-EA remarkably inhibited mushroom tyrosinase and melanogenesis in alpha-melanocyte-stimulating hormone-stimulated B16F10 cells. In addition, EME-EA significantly suppressed UVB-induced HaCaT cell death that was consistent with inhibition of apoptosis and reduction in scavenging intracellular reactive oxygen species. Furthermore, EME-EA significantly inhibited collagen degradation and matrix metalloproteinases expression in UVB-irradiated HDF cells in a concentration-dependent manner. These results indicate that EME-EA possesses strong anti-melanogenesis and photoprotective activities and suggest EME-EA is an ideal ingredient in the pharmaceutical and cosmeceutical industries.

Anti-Inflammatory Effect of Sulfated Polysaccharides Isolated from Codium fragile In Vitro in RAW 264.7 Macrophages and In Vivo in Zebrafish.

In this study, the anti-inflammatory activity of sulfated polysaccharides isolated from the green seaweed Codium fragile (CFCE-PS) was investigated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and zebrafish. The results demonstrated that CFCE-PS significantly increased the viability of LPS-induced RAW 264.7 cells in a concentration-dependent manner. CFCE-PS remarkably and concentration-dependently reduced the levels of inflammatory molecules including prostaglandin E2, nitric oxide (NO), interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-6 in LPS-stimulated RAW 264.7 cells. In addition, in vivo test results indicated that CFCE-PS effectively reduced reactive oxygen species, cell death, and NO levels in LPS-stimulated zebrafish. Thus, these results indicate that CFCE-PS possesses in vitro and in vivo anti-inflammatory activities and suggest it is a potential ingredient in the functional food and pharmaceutical industries.

Single-Cell Identification, Drug Susceptibility Test, and Whole-genome Sequencing of Helicobacter pylori Directly from Gastric Biopsy by Clinical Antimicrobial Susceptibility Test Ramanometry.

BACKGROUND: The battle against Helicobacter pylori (H. pylori) infections demands fast, reliable, and sensitive methods for pathogen identification (ID), antimicrobial susceptibility tests (ASTs) based on metabolic response, and genome-wide mutation profiling that reveals resistance mechanisms. METHODS: Here we introduce Clinical Antimicrobial Susceptibility Test Ramanometry for H. pylori (CAST-R-HP), and its validation with clinical samples. This method performs rapid ID, metabolism inhibition-based AST, and high-quality whole-genome sequencing for cells of targeted resistance phenotype, all at precisely 1-cell resolution and directly from biopsy samples. RESULTS: In CAST-R-HP, automated acquisition and machine learning of single-cell Raman spectra (SCRS) enable distinguishing individual H. pylori cells directly from a biopsy sample, with 98.5 ± 0.27% accuracy in ID. Moreover, by adding a 48- to72-h D2O feeding and drug exposure step prior to SCRS acquisition, CAST-R-HP reports AST for levofloxacin and clarithromycin with 100% accuracy, based on metabolic inhibition level. Furthermore, CAST-R-HP supports rapid sorting, low-bias DNA amplification, and full genome sequencing of single H. pylori cells with the SCRS defined, targeted drug-susceptibility phenotype, via Raman-activated gravity-driven cell encapsulation and sequencing. The genome-wide mutation map (maximum 99.70% coverage), at precisely 1-cell resolution, not only elucidates the drug-susceptibility phenotypes but also unveils their underlying molecular mechanisms. CONCLUSION: The culture independency, shorter turnaround time, high resolution, and comprehensive information output suggest that CAST-R-HP is a powerful tool for diagnosing and treating H. pylori infections.