Conventional Culture Methods Research Articles

Massive Sequencing of V3-V4 Hypervariable Region in Pyogenic Liver Abscesses Reveals the Presence of Unusual Bacteria Not Detected by Classical Culture Methods.

Pyogenic liver abscesses (PLAs) are serious infections in which doctors often fail in identifying the causative agent due to microbiological limitations. These limitations in detecting uncommon pathogens complicate the treatment and recovery. Molecular techniques, like massive sequencing, enable the detection of uncommon pathogens and highlight the shortcomings of traditional cultures. The aim of this work was to characterise the bacterial composition of PLAs through massive sequencing of the V3-V4 hypervariable region of the 16S rRNA gene in cases where conventional culture methods were negative. Purulent material was collected from three patients with PLAs at Hospital Juárez de México. Classical and molecular microbiological cultures were performed in parallel. Metagenomic DNA was extracted and massively sequenced (16S rRNA gene) using the Illumina MiSeq platform. A bioinformatic analysis was performed to determine the diversity at six different taxa levels and the relative abundances. The culture methods were not sufficient to detect the causative agent of the PLAs. However, the massive sequencing revealed the causative agents of the monomicrobial and polymicrobial infectious foci, with Gardnerella vaginalis, Lactobacillus iners, and Prevotella timonensis as the dominant bacteria. The massive sequencing revealed the presence of unusual pathogens that traditional culture failed to detect. There is an immediate need for molecular or comprehensive microbiological culture techniques to search for unusual bacteria in the diagnosis of PLAs.

Dual-stimuli-responsive nanoparticles for the co-delivery of small molecules to promote neural differentiation of human iPSCs.

The differentiation of human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs) is a promising approach for the treatment of neurodegenerative diseases and regenerative medicine. Dual-SMAD inhibition using small molecules has been identified as a key strategy for directing the differentiation of hiPSCs into NPCs by regulating specific cell signaling pathways. However, conventional culture methods are time-consuming and exhibit low differentiation efficiency in neural differentiation. Nanocarriers can address these obstacles as an efficient platform for the controlled release and accurate delivery of small molecules. In this paper, we developed calcium phosphate-coated mesoporous silica nanoparticles capable of delivering multiple small molecules, including LDN193189 as a bone morphogenetic protein (BMP) inhibitor and SB431542 as a transforming growth factor (TGF)-beta inhibitor, for direct differentiation of hiPSC-mediated NPCs. Our results demonstrated that this nanocarrier-mediated small molecule release system not only enhanced the in vitro formation of neural rosettes but also modulated the expression levels of key markers. In particular, it downregulated OCT4, a marker of pluripotency, while upregulating PAX6, a critical marker for the neuroectoderm. These findings suggest that this controlled small molecule release system holds significant potential for therapeutic applications in neural development and neurodegenerative diseases.

WORKING BODY OF A ROW CULTIVATOR

This article examines the care and maintenance of row crops, focusing on the efficiency of active and inactive working bodies of cultivators. The study provides a detailed analysis of their impact on soil aeration, weed control, and crop productivity. Particular attention is given to the design and functionality of a newly developed tool for loosening soil in the row-spacing of cotton. The innovative mechanism is designed to improve soil permeability, enhance root development, and optimize moisture retention. The article also presents preliminary research findings, highlighting the tool's effectiveness compared to conventional cultivation methods. The results indicate that the new design reduces soil compaction while maintaining the necessary structural integrity for plant growth. These insights contribute to the advancement of precision agriculture and sustainable cotton cultivation practices.

Impact of organic inputs on quality traits and nutrient content of Bottle gourd (Lagenaria siceraria L.): Heat map, PCA, correlation and path analysis

Nutrient deficiency in vegetables is a significant challenge for meeting the dietary needs of a growing population and improving the nutritional quality of bottle gourd (Lagenaria siceraria L.) through organic inputs can address this issue. An experiment was conducted at the Department of Vegetable Science, Tamil Nadu Agricultural University (2023-2024) aimed to enhance the nutritional quality of bottle gourd through organic methods. The research was conducted using a Randomized Block Design (RBD) with 11 treatment combinations and control, the study focused on improving its nutrient content, avoiding pesticide residues and supporting eco-friendly cultivation. Statistical analyses such as ANOVA, Principal Component Analysis (PCA), path analysis, phenotypic correlation and heat map analysis were used to evaluate the effects of organic inputs on the nutritional quality and market value of bottle gourd, ensuring better health benefits. Results from Season I and Season II revealed that treatment T9, which incorporated (Farmyard manure, Vermicompost, Neem cake, 3G extract, five-leaf extract, Panchagavya, Fish fermented extract, Egg fermented extract, Bacillus substilis), treatment T9 yielded the highest nutritional quality in bottle gourd, including protein (0.0275%), ascorbic acid (8.61%) and total ash (6.27%). It also had the highest macro and micronutrient levels, such as phosphorus (19.1 mg/g), potassium (1.74 mg/g), calcium (30.2 mg/g), copper (0.066 mg/g), iron (0.038 mg/g) and selenium (0.318 mg/g), showcasing its superior nutritional profile. These findings suggest that organic inputs, particularly in treatment T9, significantly enhance the nutritional quality of bottle gourd, offering a viable eco-friendly alternative to conventional cultivation methods.

Directed Cell Growth of C2C12 Cells on ECM Free Bioprinted Nano/Micro Scaffolds.

Skeletal muscle cell growth impairment can result in severe health issues, such as reduced mobility, metabolic problems, and cardiovascular issues, which can significantly impact an individual's overall health and lifestyle. To address this issue, it is essential to adopt a multi-faceted approach. Conventional 2D cell culture methods fail to replicate the critical features of in vivo micro/nanoarchitecture, which is crucial for the growth of skeletal muscle cells. In this study, the directed growth of mouse skeletal myoblasts (C2C12) cells on ECM-free biocompatible scaffolds is demonstrated and fabricated using two-photon lithography (TPL). These scaffolds are 2D and 3D and have nano/micro-features derived from chitosan-based carbon quantum dots (Ch-CQDs). Ch-CQDs act as two-photon initiators for TPL and also provide the scaffolds with adequate mechanical strength and specific binding sites. These scaffolds are biocompatible and can support cellular adhesion and growth without the need for ECM coating. The nano/micro scaffolds mimic the in vivo cellular microenvironment, enabling directed cell growth on ECM-free surfaces. The fabricated scaffolds have tunable mechanical strength ranging from 0.09 to 0.75GPa. By using Ch-CQDs, scaffolds are created that promote cell growth and alignment, which is crucial for skeletal muscle cell growth.

Customised 96-ocular TaqMan (iCAM) microarray PCR card for rapid diagnosis of microbial keratitis

AimTo validate the diagnostic performance of a custom 96-micro-organism TaqMan PCR card (iCAM) for microbial keratitis (MK) from a single corneal epithelial sample.MethodsPatients over the age of 18 referred to...

A Day Saved is a Life Saved: Direct Antimicrobial Susceptibility Testing from Positively Flagged Blood Culture Bottles and their Concordance with the Routine Method.

Sepsis is a major health problem worldwide and is associated with high morbidity and mortality with every hour delay in initiation of therapy. A conventional method of blood culture and Antimicrobial Susceptibility Testing (AST) takes around 48-72 hours. Empirical antibiotics need to be administered until the sensitivity report is made available. It has been estimated that 20-50% of the empirical antibiotics are inappropriate, resulting in prolonged hospital stays, adverse effects, and emergence of drug resistance. Additionally, this also puts an extra financial burden on both the patients and healthcare settings. Performing direct Antimicrobial Sensitivity Testing (dAST) is an important tool to reduce turn-around time (TAT) by at least 18-24 hours, thus reducing morbidity and mortality among critically ill patients. Direct AST (dAST) was performed from the positively flagged blood culture bottles received between December, 2021 to May, 2022 from Intensive Care Units (ICUs) on Mueller- Hinton Agar (MHA) using four drops of withdrawn blood. dAST was performed for six drugs: Ceftriaxone-30 μg (CTR), Piperacillin/Tazobactam-100/10 μg (PIT), Meropenem-10 μg (MRP), Ciprofloxacin-5 μg (CIP), Aztreonam-30 μg (AT), and Colistin (CL). The zone of inhibition was interpreted as per CLSI M100 ed32, 2022 guidelines. A parallel conventional method was also performed to examine for categorical agreement and disagreement. Identification was carried out using MALDI-TOF MS from the colonies that appeared on the dAST plate on the subsequent day. A total of 162 positively flagged blood culture bottles were included in the study. The majority of the Gram-negative organisms were from Enterobacterales (n=109), followed by Acinetobacter spp. (n=28) and Pseudomonas aeruginosa (n=25). Out of the 972 isolate-antimicrobial combinations, overall Categorical Agreement (CA) was seen in 936 (96.3%), whereas disagreement was observed in 36 with minor error (mE) in 21 (2.2%), major error (ME) in 7 (0.7%), and very major error (VME) in 8 (0.8%) when compared to the routine method. Categorical agreement (CA) of > 99% was seen in ceftriaxone (CTR) and ciprofloxacin (CIP). In comparison, the lowest CA was observed with meropenem (MRP) at 92%. Colistin dAST was performed using the E-strip method, and the result obtained was highly convincing, with an overall disagreement of only 1.2%. Rapid dAST from positively flagged blood culture bottles proved to significantly reduce the TAT from the time of sample collection to the first availability of antimicrobial susceptibility report with excellent categorical agreement of > 95% using the conventional disc diffusion method. Results obtained were within the acceptance criteria set by U. S. Food and Drug Administration (FDA) guidelines of > 90% categorical agreement for a new method. We were able to obtain excellent concordance for colistin using the E-strip method. Performing dAST not only saves a "day", but its proper implementation would save a "life".

Advancements in Organoid Culture Technologies: Current Trends and Innovations.

Organoids have emerged as valuable tools in investigating disease mechanisms, drug efficacy, and personalized medicine due to their capacity to recapitulate crucial aspects of tissue physiology, including cell-cell interactions, heterogeneity, microenvironmental cues, and drug responses. Despite their broad applicability across various research domains, conventional organoid culture methods are plagued by several limitations that hinder research progress. These limitations include the inability to faithfully recreate tissue microenvironments, immune contexts, and vascular systems. Fortunately, ongoing advancements in organoid culture techniques are addressing these shortcomings. In this review, we provide a comprehensive overview of current mainstream organoid culture protocols. By evaluating these protocols, researchers can identify the most suitable experimental methods, thereby optimizing resource allocation and experimental outcomes.

Large, protracted, multi-species and multi-clonal spread of VIM-type metallo-β-lactamase-producing Enterobacterales in an Italian hospital.

Carbapenem-resistant Enterobacterales, particularly those producing carbapenemase (CPE), pose a major threat to human health, being listed among critical-priority resistant pathogens by the World Health Organization. To report on a large nosocomial spread of CPE of different species producing Verona integron-encoded metallo-β-lactamase (VIM)-type carbapenemases, and on the infection prevention and control measures that were adopted to combat the spread. Conventional culture and molecular methods were used for detection and identification of VIM-positive CPE (VIM-CPE) causing infections or colonizing patients or present in environmental specimens. Whole-genome sequencing analysis of selected isolates was performed to investigate clonal relatedness. Basic (active surveillance, contact precautions, close contact screening, cohorting of patients, surface cleaning, hand hygiene) and advanced (weekly point-prevalence surveys for rectal colonization, additional training of healthcare workers, extraordinary ward sanitization, extraordinary maintenance interventions, and environmental microbiological screening, single-use equipment, ward relocation) infection prevention and control (IPC) measures were implemented to combat the spread. Spread of VIM-CPE involving 151 patients (mostly colonizations) was documented in a single hospital ward from November 2021 to December 2023. The spread involved several different species of Enterobacterales, with clonal expansion documented in some cases. Implementation of basic and advanced IPC measures was temporarily successful at mitigating the spread, but multiple relapses were observed, suggesting the presence of an unidentified environmental reservoir. VIM-CPE has the potential to cause large and complex nosocomial outbreaks in hospital environments, underscoring the challenges to their control by IPC practices.

Analysis of water and aerosol samples of tunnel car washes operated with recycled water for Legionella with culture, qPCR and viability-qPCR

Due to the generation of large quantities of aerosol and the recycling of water, tunnel car washes are discussed as potential sources of legionellosis. Additionally, occupational health and safety aspects are important for tunnel car washes as they are often workplaces.A total of 17 different tunnel car washes were investigated for the presence of Legionella. In the process, 78 water samples and 63 air samples were taken. This comprised samples of municipal and recycled water as well as aerosol from the car wash tunnel and the workplace. The analysis for Legionella included culture method in combination with an immunochromatographic test, qPCR and viability-qPCR. Where possible, Legionella species were identified by sequencing of mip gene.Using the culture method Legionella were detected in 9 of 78 water samples (91 to 10,800 CFU/100 mL). In contrast, quantifiable concentrations of viable Legionella spp. were found in 68 of 78 water samples with viability-qPCR. The median concentration was 9.2 × 105(n = 16) and 7.2 × 105 GU/100 mL (n = 17) for the recycled water from the storage tank and the nozzles in the car wash tunnel. Viable Legionella spp. were detectable in the aerosol at the workplace in 38.1 % of the samples (n = 21). Concentration was between 155 and 3829 GU/m3 (n = 7). L. pneumophila non-serogroup 1 were quantitatively detectable in the recycled water of one car wash, using qPCR methods and culture. Aerosolisation of this species was not detected.The presence of viable Legionella spp. in most water and many aerosol samples as well as the identification of species related to infection suggests that there is a risk of legionellosis through exposure to bioaerosols released from tunnel car washes. Comparison of conventional culture method with qPCR methods showed a considerable underestimation of Legionella concentrations by culture.

Identification of fungal pathogens among COVID-19 and non COVID-19 cases in Bhaktapur hospital, Nepal

ObjectivesPatients with coronavirus disease 2019 (COVID-19) are at increased risk of opportunistic fungal infections. This study aims to identify fungal pathogens among COVID positive and negative patients, assess their antifungal susceptibility and evaluate biofilm forming ability of Candida spp. A cross-sectional study was conducted among sputum samples from 135 COVID positive and 101 COVID negative cases. Fungal pathogens were identified by conventional culture methods. Antifungal susceptibility test of Candida isolates was done by disc diffusion method and biofilm production by microtiter plate method.ResultsThe prevalence of fungal pathogens among COVID-positive and negative cases was 6.70% and 22.77% respectively. In COVID positive cases, Candida albicans (33.33%) was predominantly followed by Aspergillus flavus 2(22.22%) and Candida tropicalis, Mucor spp. and Aspergillus fumigatus. In COVID negative cases, Candida albicans (69.60%) prevailed followed by Trichosporon spp., Candida parapsilosis, Mucor and Alternaria. Age and gender were not associated with fungal infection. Most Candida spp. were susceptible to miconazole but resistant to ketoconazole. To the best of our knowledge, this study represents the first report from Nepal on critical and high priority fungal pathogens categorized by WHO. With fungal infections on the rise, enhanced clinical vigilanceand antifungal susceptibility testing are warranted.

Method for Identification and Bacterial Count Quantification in a Case of Ureaplasma Meningitis.

Intrauterine Ureaplasma infection is associated with chorioamnionitis and preterm birth. The difficulty of detecting Ureaplasma species by conventional culture methods makes definitive diagnosis of clinical infection challenging. Thus far, quantitative tests for Ureaplasma have been performed using adult cervical samples, amniotic fluid, and pediatric bronchial secretions, but quantification of bacterial count in central nervous system infections caused by Ureaplasma species has not been unreported. We report a case of culture-negative Ureaplasma meningitis in a preterm infant in whom novel techniques to identify this pathogen and quantify bacterial count were effective. We suspected meningitis based on a sustained reduction in cerebrospinal fluid (CSF) glucose levels. Multiple CSF cultures were sterile. We confirmed infection by Ureaplasma species using the melting temperature mapping method. Treatment with erythromycin and ciprofloxacin resulted in a gradual decrease in the bacterial count in the CSF to 0. Our study highlights the potential utility of the melting temperature mapping method as a new diagnostic tool for culture-negative Ureaplasma meningitis and establishes the utility of serial quantification of bacterial count to monitor response to therapy.

Performance of a hybrid capture-based target enrichment next-generation sequencing for the identification of respiratory pathogens and resistance-associated genes in patients with severe pneumonia.

Severe pneumonia remains the leading infectious cause of death worldwide. The time-consuming nature and suboptimal sensitivity of sputum cultures hamper prompt pathogen detection for tailored treatments. Advanced techniques such as polymerase chain reaction (PCR) and next-generation sequencing (NGS) offer rapid genetic pathogen detection and identification of antimicrobial resistance (AMR) genes. However, the performance of hybrid capture-based target enrichment NGS, e.g., Respiratory Pathogen ID/AMR Enrichment Panel (RPIP), for pathogen detection in patients with severe pneumonia remains uncertain. A prospective study involving adults with severe pneumonia was conducted. Respiratory samples from the lower respiratory tract were collected via bronchoalveolar lavage, bronchial washing, or endotracheal tube suction. The performance of RPIP in pathogen and AMR-associated gene detection was compared to that of conventional culture methods and the multiplex PCR-based FilmArray Pneumonia Panel (FilmArray-PN). A total of 83 subjects were enrolled. The most prevalent pathogens detected by RPIP were Rothia mucilaginosa, Stenotrophomonas maltophilia, Pseudomonas aeruginosa; herpes simplex virus-1, cytomegalovirus, and Epstein-Barr virus, and Pneumocystis jirovecii. Overall, the positive and negative agreement rates for bacterial detection were 63.6% and 97.5% between RPIP and culture methods, respectively, and 55.8% and 99.4% between FilmArray-PN and culture methods, respectively. Compared to FilmArray-PN, RPIP exhibited significantly better detection rates for bacteria (P = 0.029), viruses (P < 0.001), and fungi (P < 0.001) and identified additional blaOXA, blaCMY as extended-spectrum β-lactamase genes and blaOXA, blaSHV as carbapenemase genes. In conclusion, RPIP can sensitively profile respiratory pathogens and is a promising tool for detecting multiple microorganisms and AMR-associated genes in patients with severe pneumonia.IMPORTANCESensitive pathogen detection is pivotal for timely treatment by tailoring adequate antimicrobial agents. Unlike conventional phenotypic approach, novel measures using molecular interrogation appear promising. This study aimed to elucidate the efficacy of a hybrid capture-based target enrichment next-generation sequencing technique (Respiratory Pathogen ID/AMR Enrichment Panel, RPIP) as exemplified in a cohort with severe pneumonia. Pathogen landscape in the population was illustrated by these three methodologies. As compared with multiplex polymerase chain reaction-based FilmArray Pneumonia Panel and conventional culture, RPIP demonstrated significantly improved sensitivity in identifying bacteria, viruses, and fungi. The RPIP also exhibited better performance in identifying different pathogens in patients co-infected with multiple microorganisms. Additionally, the genotypes contributing to antimicrobial resistance were determined by RPIP. The study facilitated the implementation of molecular diagnosis by presenting real-world data, whereas future studies are mandated to generalize such an approach toward different clinical settings.

Temporal dynamics of Escherichia coli O157:H7 transcriptomes on the surface of shredded lettuce

Escherichia coli (E. coli) O157:H7 is one of the major foodborne pathogens associated with lettuce-related outbreaks over recent decades. Our study illustrates that this pathogen not only survives on the lettuce surface but also undergoes replication and metabolic alterations. Conventional culture methods showed approximately 2.5 log cfu/g increase in the E. coli O157:H7 population on lettuce, from 5.27 log cfu/g to 7.71 log cfu/g without any external energy source. Our transcriptome study revealed 1003 differentially expressed genes (DEGs) (|log2FoldChange|>2 and Padj < 0.05) after 1 h storage and 355 DEGs after 18 h of storage. The majority of upregulated genes observed at the 1-h storage lettuce compared with pure culture, were associated with metabolism, biosynthesis of major amino acids (such as methionine, tryptophan, arginine, and branched-chain amino acids), and stress response. Conversely, genes upregulated in the 18 h of storage lettuce samples, compared with the 1-h samples, were linked to anaerobic respiration, biosynthesis of alternative amino acids, and surface attachment. The downregulation of metabolism and amino acid biosynthesis genes was observed in these samples. Additional analysis mapped the DEGs to metabolic pathways using the DAVID database, and showed most genes were clustered with metabolism and ion transport pathways. This investigation identified DEGs involved in the initial adaptation phase and the growth of E. coli O157:H7 during storage on the lettuce surface, shedding light on the molecular mechanisms underlying its survival and proliferation in this environment.

Improvement of Transplanting Rice Yield and Nitrogen Use Efficiency by Increasing Planting Density in Northeast China Under the Optimal Nitrogen Split-Fertilizer Applications

There are few studies on how nitrogen (N) fertilizer application rates and transplanting densities impact rice yield, root distribution, and N use efficiency in the cold regions of Northeast China. This research involved a two-year field trial utilizing Jinongda 667 as the material. In 2021, three N split-fertilizer applications—T1 (6:3:1), T2 (5:3:2), T3 (4:3:3)—and two transplanting densities—D1 (30 cm × 13.3 cm) and D2 (30 cm × 20 cm)—were compared with the conventional cultivation mode (T0: 175 kg N hm−2, 6:3:1), whereby the N application mode most suitable for increasing density was explored. In 2022, four N application levels—0 (N0), 125 (N1), 150 (N2), and 175 (N3) kg N hm−2—were assessed under the same density treatment to analyze the yield, resource utilization efficiency, and root traits of Jinongda 667. The results indicated that when the transplanting density was 30 cm × 13.3 cm, the application of 5:3:2 fertilizer was more conducive to improving rice yield. Increasing planting density under reduced N input significantly enhanced both rice yield and N use efficiency. In contrast to the conventional cultivation method (D2N3), the treatment of increased planting density (D1N2) under reduced N input led to a 21.2% rise in the number of panicles per square meter and an 8.6% boost in rice yield. Furthermore, increasing planting density under reduced N input significantly enhanced the agronomic efficiency of N fertilizer, the apparent utilization rate, and the N harvest index. It also boosted the SPAD value, photosynthetic rate, and the utilization efficiency of light and N resources in rice. However, it was noted that root enzyme activity decreased. This study demonstrated that increasing planting density, combined with the N application mode of 5:3:2 and an N application rate of 150 kg hm−2, maximized resource utilization efficiency, optimized root absorption capacity, and resulted in higher yields.

Epidemiology of pediatric meningitis and encephalitis in Japan: a cross-sectional study.

Information on the epidemiology and cause-specific clinical features of pediatric meningitis and encephalitis is limited, owing to conventional laboratory methods' limitations in identifying causative pathogens. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a molecular diagnostic tool, can detect 14 pathogens within 1 hour. We investigated meningitis and encephalitis epidemiology among children in Japan and FA-M/E panels' utility in their clinical management. This cross-sectional study was conducted among children aged 0-18 years admitted to seven regional hospitals in western Japan between October 2022 and September 2023. Cerebrospinal fluid (CSF) samples were collected from 221 children and tested using the FA-M/E panel. Clinical and microbiological data were reviewed retrospectively. Fifty-eight patients tested positive using the FA-M/E panel. Viral and bacterial pathogens were detected in 49 and nine cases, respectively. Human parechovirus and enterovirus occurred mainly in epidemic clusters during the summer and were primarily detected in young infants. Patients who tested positive for human parechovirus had a significantly higher frequency of sepsis-like manifestations and a lower frequency of CSF pleocytosis than did those who tested positive for enterovirus. CSF pleocytosis was absent in 30 patients who tested positive using the FA-M/E panel. Among the patients who tested positive for bacteria, three of the nine were not diagnosed using conventional culture methods, owing to prior antimicrobial therapy. The FA-M/E panel can identify bacterial and viral pathogens causing pediatric meningitis and characterize the epidemiology in local communities.IMPORTANCECulture and polymerase chain reactions have traditionally been used to identify microorganisms causing pediatric meningitis and encephalitis. However, the methods currently used to identify the causative microorganisms are limited, particularly in general hospitals. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a fully automated genetic testing system, can detect 14 pathogens using the multiplex polymerase chain reaction method. This study described the epidemiology of pediatric meningitis and encephalitis in Japan. The microorganisms causing acute meningitis and encephalitis in children in Japan were identified using the FilmArray Meningitis/Encephalitis panel. Testing cerebrospinal fluid using the FA-M/E panel is useful for the identification of the pathogen in children with community-acquired acute meningitis and encephalitis. This increases knowledge on the epidemiology and clinical manifestations of acute meningitis and encephalitis caused by specific pathogens and can be used to facilitate optimal patient management.

High utility of bronchoalveolar lavage fluid metagenomic next-generation sequencing approach for etiological diagnosis of pneumonia

BackgroundFor patients with pneumonia, the rapid detection of pathogens is still a major global problem in clinical practice because traditional diagnostic techniques for infection are time-consuming and insensitive. Metagenomic next-generation sequencing (mNGS) is a novel technique that has the potential to improve pathogen diagnosis. This study aimed to investigate the microbiological diagnostic ability of mNGS compared with conventional culture and to determine the optimal time to test patients for pneumonia.MethodsA prospective study using data from June 2020 to June 2021 was performed at a tertiary teaching hospital in China. We included 56 patients from all adult patients with a clinical diagnosis of pneumonia. Blood and bronchoalveolar lavage fluid (BALF) samples were taken for simultaneous mNGS and conventional culture testing.ResultsAll 56 patients underwent both conventional culture and mNGS. Of these patients, 37 were diagnosed with severe pneumonia and 17 were diagnosed with non-severe pneumonia. The top three pathogenic bacteria detected by mNGS were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Enterococcus faecium was detected more frequently in the non-severe pneumonia group (4 vs. 0, p < 0.05). The findings revealed that the detection rate of mNGS (84%) was superior to that of conventional culture methods (48%). Notably, the percentage of mNGS-positive BALF samples (46/56, 82.14%) was significantly greater than that of blood samples (27/56, 48.21%). The etiological comparison demonstrated that mNGS-positive samples, which received clinical approval, tended to be associated with a more normalized temperature, lower PCO2 levels, and a higher SOFA score than mNGS-negative samples (p = 0.022, p = 0.0.028, and p = 0.038, respectively).ConclusionsIn this study, we discovered that the etiology of lung infections frequently involves multiple pathogens. The use of mNGS in BALF is instrumental for detecting nonviral pathogens associated with lung infections. Although the rate of positive blood NGS results is significantly influenced by various clinical factors, for patients suspected of having viral, Legionella, or tsutsugamushi infections, plasma mNGS could serve as a complementary diagnostic tool.

Study on the Structure and Function of Intestinal Microorganisms in Silkworm Maggot Exorista sorbillans

ABSTRACTInsects have important symbiotic relationships with their intestinal microbiota. The intestinal microbiota is involved in or influences various processes in insects such as development, metabolism, immunity, and reproduction. Currently, research on the intestinal microbiota of parasitic insects is still in its early stages. The tachinid parasitoid Exorista sorbillans is a dipteran parasitic insect, with the silkworm (Bombyx mori) being its main host. Silkworms parasitized by E. sorbillans can suffer from severe silkworm maggot disease, which also poses a serious threat to sericulture. In this study, the intestinal microbiota of larval E. sorbillans at three instar stages was analyzed using 16S rRNA amplicon sequencing to explore the community composition of the intestinal microbiota. Additionally, using conventional culture methods, six cultivable strains were isolated and identified from the larval E. sorbillans on an antibiotic‐free LB medium, and four cultivable strains were isolated and identified from the hemolymph of parasitized silkworms. This study investigated the E. sorbillans from the perspective of intestinal microbiota, elucidating the composition and structural characteristics of the intestinal microbiota of the tachinid parasitoid, and preliminarily discussing the functional roles of several major microorganisms, which helps to further clarify the potential mechanisms of interaction between the parasitoid and the silkworm.

Assessing the Impact of Climate Change on Methane Emissions from Rice Production Systems in Southern India

The impact of climate change on methane (CH4) emissions from rice production systems in the Coimbatore region (Tamil Nadu, India) was studied by leveraging field experiments across two main treatments and four sub-treatments in a split-plot design. Utilizing the closed-chamber method for gas collection and gas chromatography analysis, this study identified significant differences in CH4 emissions between conventional cultivation methods and the system of rice intensification (henceforth SRI). Over two growing seasons, conventional cultivation methods reported higher CH4 emissions (range: from 36.9 to 59.3 kg CH4 ha−1 season−1) compared with SRI (range: from 2.2 to 12.8 kg CH4 ha−1 season−1). Experimental data were subsequently used to guide parametrization and validation of the DeNitrification–DeComposition (DNDC) model. The validation of the model showed good agreement between the measured and modeled data, as denoted by the statistical tests performed, which included CRM (0.09), D-index (0.99), RMSE (7.16), EF (0.96), and R2 (0.92). The validated model was then used to develop future CH4 emissions projections under various shared socio-economic pathways (henceforth SSPs) for the mid- (2021–2050) and late (2051–2080) century. The analysis revealed a potential increase in CH4 emissions for the simulated scenarios, which was dependent on specific soil and irrigation management practices. Conventional cultivation produced the highest CH4 emissions, but it was shown that they could be reduced if the current practice was replaced by minimal flooding or through irrigation with alternating wetting and drying cycles. Emissions were predicted to rise until SSP 370, with a marginal increase in SSP 585 thereafter. The findings of this work underscored an urgency to develop climate-smart location-specific mitigation strategies focused on simultaneously improving current water and nutrient management practices. The use of methanotrophs to reduce CH4 production from rice systems should be considered in future work. This research also highlighted the critical interaction that exists between agricultural practices and climate change, and emphasized the need to implement adaptive crop management strategies that can sustain productivity and mitigate the environmental impacts of rice-based systems in southern India.

Unveiling the Human Brain on a Chip: An Odyssey to Reconstitute Neuronal Ensembles and Explore Plausible Applications in Neuroscience.

The brain is an incredibly complex structure that consists of millions of neural networks. In developmental and cellular neuroscience, probing the highly complex dynamics of the brain remains a challenge. Furthermore, deciphering how several cues can influence neuronal growth and its interactions with different brain cell types (such as astrocytes and microglia) is also a formidable task. Traditional in vitro macroscopic cell culture techniques offer simple and straightforward methods. However, they often fall short of providing insights into the complex phenomena of neuronal network formation and the relevant microenvironments. To circumvent the drawbacks of conventional cell culture methods, recent advancements in the development of microfluidic device-based microplatforms have emerged as promising alternatives. Microfluidic devices enable precise spatiotemporal control over compartmentalized cell cultures. This feature facilitates researchers in reconstituting the intricacies of the neuronal cytoarchitecture within a regulated environment. Therefore, in this review, we focus primarily on modeling neuronal development in a microfluidic device and the various strategies that researchers have adopted to mimic neurogenesis on a chip. Additionally, we have presented an overview of the application of brain-on-chip models for the recapitulation of the blood-brain barrier and neurodegenerative diseases, followed by subsequent high-throughput drug screening. These lab-on-a-chip technologies have tremendous potential to mimic the brain on a chip, providing valuable insights into fundamental brain processes. The brain-on-chip models will also serve as innovative platforms for developing novel neurotherapeutics to address several neurological disorders.