Microbial Samples Research Articles

Toxic buffers used in microbial sample preservation for SEM can be replaced by PBS

Toxic buffers used in microbial sample preservation for SEM can be replaced by PBS

Metagenomic 18S rDNA reads revealed zonation of eukaryotic communities in the Yongle blue hole.

The Yongle blue hole (YBH), situated in the South China Sea, represents a compelling subject of study in marine microbiology due to its unique redox-layered microbial ecosystems. However, the diversity and ecology of microbial eukaryotes within the YBH remains underexplored. This study endeavors to bridge this gap through the application of the in situ microbial filtration and fixation (ISMIFF) device to collect 0.22-30 μm microbial samples from 21 water layers of YBH. Subsequent extraction of 18S rRNA metagenomic reads of 21 metagenomes and 10 metatranscriptomes facilitated a comprehensive analysis of community structures. Findings revealed a pronounced superiority in the diversity and richness of eukaryotic microorganisms in the oxic zone compared to its suboxic and anoxic counterparts. Notably, Dinophyceae and Maxillopoda emerged as the predominant taxa based on the analysis of the 18S rRNA reads for the V4 and V9 regions, which showed stratification In their relative abundance and suggested their potential role in the thermo-halocline boundaries and oxic-anoxic interface. Specifically, In these eukaryotic microbial communities, Dinophyceae exhibited significant abundance at 20 m (20.01%) and 105 m (26.13%) water depths, while Maxillopoda was prevalent at 40 m (22.84%), 80 m (23.19%), and 100 m (15.42%) depths. A part of these organisms, identified as larvae and protists, were likely attracted by swarming chemosynthetic bacterial prey prevailing at the thermo-halocline boundaries and oxic-anoxic interface. Furthermore, the phylogenetic relationships of the major 18S operational taxonomic units (OTUs) showed a close adjacency to known species, except for three Dinophyceae OTUs. In conclusion, this study provides critical insights into the vertical distribution and transcriptional activity of <30-μm eukaryotic microbes, shedding light on the taxonomic novelty of eukaryotic microorganisms within the semi-enclosed blue holes.

The role of probiotics for preventing dysbiosis in periodontal disease: a randomized controlled trial.

Scaling and root planing remain inadequate in periodontitis treatment caused by dysbiotic microbial dental plaque. The aim of this clinical trial is to evaluate the effects of probiotics and kefir consumption in initial periodontal therapy (IPT) on oral microbiota composition and treatment outcomes in patients with periodontitis. The study was carried out in the Gazi University Department of Periodontology, including a sample size of 36 individuals and utilizing a randomized controlled design. Thirty-six patients with periodontitis were randomly allocated to three groups: one receiving probiotic treatment, another receiving kefir, and a third serving as the control group. Obtaining subgingival microbial samples, we recorded plaque, gingival index, bleeding on probing, periodontal pocket depth, and clinical attachment level (periodontal clinical indices) and then performed IPT. For 14 days, patients took either probiotics, kefir, or no supplements. Data for the first and third months were collected using periodontal clinical indices. DNA sequencing was performed to detect Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola in subgingival plaque samples collected at baseline and three months. Significant differences were observed regarding periodontal clinical indices among groups in the intragroup comparisons. Moreover, levels of Tannerella forsythia were significantly decreased in all groups. Kefir can be administered in addition to IPT, providing results similar to those observed with probiotics.

Proteomic Analysis of HepG2 Cells Reveals FAT10 and BAG2 Signaling Pathways Affected by a Protease Inhibitor from Tinospora cordifolia (Willd.) Hook. f. and Thoms Stem. Extract Among the Different Plant and Microbial Samples Analyzed.

Dysregulation of proteolysis underlies diseases like cancer. Protease inhibitors (PIs) regulate many biological functions and hence have potential anticancer properties. With this background, the current study aimed to identify the PI from natural sources such as plants and microbes against trypsin (a protease), which was assayed against casein, using an ultraviolet spectrophotometer-based methodology. PI extracted from a few plants and microbial samples were screened for their PI activity against trypsin. The PI from the most promising source in our study, Tinospora cordifolia (Willd.) Hook. f. and Thoms. stem, was partially purified using ammonium sulfate precipitation followed by dialysis. The PI activity of the partially purified inhibitor was analyzed against chymotrypsin and collagenase enzymes, and the cytotoxic effect of the PI was checked on HepG2 (liver carcinoma) cells by MTT- [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]- assay. Liquid Chromatograography Mass Spectrometry -based proteomic studies were performed on HepG2 cells to understand the signaling pathways affected by the PIs in the liver cancer cell line. Among the samples tested the PIs from T. cordifolia stem extract had the highest inhibitory activity (72.0%) against trypsin along with cytotoxicity to HepG2 cells. After partial purification by 80.0% ammonium sulfate precipitation, PI had increased inhibitory activity (83.0%) against trypsin and enhanced cytotoxicity (47.0%) to HepG2 cells. Proteomic analysis of the PI-treated HepG2 cells revealed that BAG2 and FAT10 signaling pathways were affected, which may have caused the inhibition of cancer cell proliferation. PI from T. cordifolia stem has promising anticancer potential and hence can be used for further purification and characterization studies toward cancer drug development.

Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay

C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.Graphical

Experimental study on the influence mechanism of clay particles on the microbial treatment of granite residual soil

The content of clay particles has a key influence on the physical and mechanical properties of microbial solidified granite residual soil. Microbial-induced calcite precipitation (MICP) treatment method of “mixing + grouting” was used to prepare microbial solidified granite residual soil samples with different clay contents and to explore the influence of clay content on the reinforcement effect of microbial solidified granite residual soil. A series of direct shear tests, disintegration tests, and microscopic observation tests was conducted to quantitatively analyze the influence of clay content on the shear strength and disintegration resistance of microbial solidified granite residual soil. The conclusions are summarized as follows: the shear strength and disintegration resistance of granite residual soils solidified by MICP treatment were remarkably improved, with the shear strength increasing by 9%∼16% when the clay content varied from 10%∼40%. The internal friction angle of the granite residual soil with different clay contents increased and then decreased with the increase in clay content. The cohesion of the microbial solidified granite residual soil increased with the increase in clay content, and the internal friction angle reached a minimum of 20% clay content (17.27°), which is 31.85% lower than that of 0% clay content (25.34°). The cohesion of the microbial solidified granite residual soil reached a maximum of 40% clay content (39.54 kPa), which is 8.69 times higher than that of 0% clay content (4.55 kPa). The MICP treatment technology is capable of effectively improving the shear strength and disintegration resistance of granite residual soils. The MICP treated process produces calcium carbonate precipitates that link the sand particles and fill the pores between the soil particles, increasing the number of larger agglomerates in the granite residual soil, which is the essential reason for the improvement in the softening and disintegration of the granite residual soil when exposed to water.

Optimized sample buffer for dispersed, high-resolution capillary zone electrophoretic separation of Escherichia coli B

Capillary zone electrophoresis (CZE) is a powerful tool for high resolution chemical separations. Applying CZE to microbial samples may facilitate a deeper understanding of bacterial physiology and behavior. However, the study of complex microbial samples has been limited by the uncontrolled hetero-aggregation of bacterial cells under an applied electric field. We tested a wide range of sample buffers and buffer additives for the optimization of bacterial CZE separations using a 20 mM Tris–HCl background electrolyte. By modifying the sample buffer, but not the background electrolyte, we retain constant separation conditions, which aids in the comparison of the sample buffer additives. We report optimized methods for automated CZE separation and simultaneous fractionation of Escherichia coli B, which is one of the two most widely used wild-type strains. A modified sample buffer containing neutral salts and the addition of glycerol produced a 20-fold increase in loading capacity and a reduction in peak width/broadening of 86% in comparison to previously reported work. In addition, the glycerol-modified sample buffer appears to reduce the persistent aggregation and adhesion to the capillary walls during electrophoretic separations of complex environmental microbiota.

Effect on the oxidation characteristic at lignite of two types of endogenous microorganisms from coal

In recent years, coal biotechnology has gained considerable popularity due to its economic, eco-friendly, and sustainable advantages, which offer a novel perspective and pathway for the development of green technologies aimed at preventing and controlling coal spontaneous combustion (CSC). Two endogenous microorganisms from lignite were succeeded in extracting in this work, namely Aspergillus and Bacillus velezensis. Subsequently, the effect of these microorganisms on the microscopic structure and macroscopic oxidation characteristics of coal was investigated by X-ray diffraction, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The results revealed that both Aspergillus and Bacillus velezensis treatments altered the microcrystalline structure of the coal, disrupting the aliphatic hydrocarbon structure and oxygen-containing functional groups of the coal molecules. Particularly, the OH − O hydrogen bond of two kinds of microbial treatment coal samples content respectively decreased significantly by 17.29 % and 11.06 % compared to the raw coal sample. However, the effects of different microorganisms on coal varied. Thermodynamic behavior demonstrated that the critical temperature of the two treated coal samples was delayed by 29.44 °C and 26.62 °C, and the ignition temperature was delayed by 11.31 °C and 5.18 °C. Overall, all characteristic temperature points of the Aspergillus group were delayed, but the maximum weight loss rate temperature and the burn-out temperature of the Bacillus velezensis group exhibited slight advancement. The thermal mass loss characteristics also changed during the oxidation combustion stage. In addition, the average activation energies of oxidized combustion stage calculated by the Kissinger-Akahira-Sunose method were 74.26 kJ/mol and 78.61 kJ/mol, respectively, and 82.41 kJ/mol and 86.15 kJ/mol for the Friedman method, which were both increased compared to the raw coal. The results indicate that both Aspergillus and Bacillus velezensis have effects with varying degrees on the coal oxidation characteristics. It can be observed that the effect of treatment with Aspergillus is better than Bacillus velezensis from the overall changes in characteristic temperature points. This work lays the foundation for investigating the effect of microbial action on the oxidative properties of CSC.

Metabolomics of natural samples: A tutorial review on the latest technologies.

Metabolomics is the study of metabolites present in a living system. It is a rapidly growing field aimed at discovering novel compounds, studying biological processes, diagnosing diseases, and ensuring the quality of food products. Recently, the analysis of natural samples has become important to explore novel bioactive compounds and to study how environment and genetics affect living systems. Various metabolomics techniques, databases, and data analysis tools are available for natural sample metabolomics. However, choosing the right method can be a daunting exercise because natural samples are heterogeneous and require untargeted approaches. This tutorial review aims to compile the latest technologies to guide an early-career scientist on natural sample metabolomics. First, different extraction methods and their pros and cons are reviewed. Second, currently available metabolomics databases and data analysis tools are summarized. Next, recent research on metabolomics of milk, honey, and microbial samples is reviewed. Finally, after reviewing the latest trends in technologies, a checklist is presented to guide an early-career researcher on how to design a metabolomics project. In conclusion, this review is a comprehensive resource for a researcher planning to conduct their first metabolomics analysis. It is also useful for experienced researchers to update themselves on the latest trends in metabolomics.

Core gut microbes Cloacibacterium and Aeromonas associated with different gastropod species could be persistently transmitted across multiple generations

BackgroundStudies on the gut microbiota of animals have largely focused on vertebrates. The transmission modes of commensal intestinal bacteria in mammals have been well studied. However, in gastropods, the relationship between gut microbiota and hosts is still poorly understood. To gain a better understanding of the composition of gut microbes and their transmission routes in gastropods, a large-scale and long-term experiment on the dynamics and transmission modes of gut microbiota was conducted on freshwater snails.ResultsWe analyzed 244 microbial samples from the digestive tracts of freshwater gastropods and identified Proteobacteria and Bacteroidetes as dominant gut microbes. Aeromonas, Cloacibacterium, and Cetobacterium were identified as core microbes in the guts, accounting for over 50% of the total sequences. Furthermore, both core bacteria Aeromonas and Cloacibacterium, were shared among 7 gastropod species and played an important role in determining the gut microbial community types of both wild and cultured gastropods. Analysis of the gut microbiota at the population level, including wild gastropods and their offspring, indicated that a proportion of gut microbes could be consistently vertically transmitted inheritance, while the majority of the gut microbes resulted from horizontal transmission. Comparing cultured snails to their wild counterparts, we observed an increasing trend in the proportion of shared microbes and a decreasing trend in the number of unique microbes among wild gastropods and their offspring reared in a cultured environment. Core gut microbes, Aeromonas and Cloacibacterium, remained persistent and dispersed from wild snails to their offspring across multiple generations. Interestingly, under cultured environments, the gut microbiota in wild gastropods could only be maintained for up to 2 generations before converging with that of cultured snails. The difference observed in gut bacterial metabolism functions was associated with this transition. Our study also demonstrated that the gut microbial compositions in gastropods are influenced by developmental stages and revealed the presence of Aeromonas and Cloacibacterium throughout the life cycle in gastropods. Based on the dynamics of core gut microbes, it may be possible to predict the health status of gastropods during their adaptation to new environments. Additionally, gut microbial metabolic functions were found to be associated with the adaptive evolution of gastropods from wild to cultured environments.ConclusionsOur findings provide novel insights into the dynamic processes of gut microbiota colonization in gastropod mollusks and unveil the modes of microbial transmission within their guts.1dB18pPYxP-ygVgUJj2q9BVideo

Bacterial dynamic of flue-cured tobacco leaf surface caused by change of environmental conditions

Microorganisms present on the surface of tobacco leaves play a significant role in shaping the composition of the tobacco microbial ecosystem, which undergoes continuous changes throughout the curing process. In the present study, a total of four distinct tobacco curing periods were selected for sampling, namely the fresh, yellowing, leaf-drying, and stem-drying stages. The bacterial 16S rRNA gene sequences of the collected samples were subsequently analyzed to identify operational taxonomic units (OTUs). The findings indicated that the complete dataset of leaf microbial samples was clustered, resulting in the identification of 1,783 operational taxonomic units (OTUs). Furthermore, the analysis of diversity revealed a pattern of initially increasing and subsequently decreasing community diversity. Redundancy Analysis (RDA) and weighted gene correlation networks for analysis (WGCNA) were employed in conjunction with environmental factors to assign OTUs to 22 modules for functional analysis. Additionally, a classification model utilizing the random forest algorithm was utilized to identify seven marker microorganisms (Escherichia coli, Faecalibacterium prausnitzii, Faecalibacterium, Escherichia-Shigella, Peptostreptococcaceae, Peptostreptococcales-Tissierellales, and Proteobacteria) that exhibited discriminative characteristics across different time periods. This study aimed to investigate the dynamic changes in the bacterial community throughout the curing process and their impact on the community’s function. Additionally, certain bacteria were identified as potential markers for detecting changes in the curing stage. These findings offer a novel opportunity to accurately regulate the curing environment, thereby enhancing the overall quality of tobacco leaf curing.

Simultaneous nitrate and chromium removal mechanism in a pyrite-involved mixotrophic biofilter.

Microbially mediated NO3--N and Cr(VI) reduction is being recognized as an eco-friendly and cost-effective remediation strategy. Iron sulfide mineral, as a natural inorganic electron donor, has a strong influence on NO3--N and Cr(VI) transformation, respectively. However, little is known about the simultaneous nitrate and chromium removal performance and underlying mechanism in an iron sulfide mineral-involved mixotrophic biofilter. This study demonstrated that the NO3--N and Cr(VI) removal efficiencies were stable at 62 ± 8% and 56 ± 10%, and most of them were eliminated in the 0-100-mm region of the biofilter. Cr(VI) was reduced to insoluble Cr(III) via microbial and chemical pathways, which was confirmed by the SEM-EDS morphology and the XPS spectra of biofilm and pyrite particles. SO42- was as a main byproduct of pyrite oxidation; however, the bacterial SO42- reduction synchronously occurred, evidenced by the variations of TOC and SO42- concentrations. These results suggested that there were complicated and intertwined biochemical relations between NO3--N/Cr(VI)/SO42-/DO (electron acceptors) and pyrite/organics (electron donors). Further investigation indicated that both the maximal biomass and greatest denitrifiers' relative abundances in microbial sample S1 well explained why the pollutants were removed in the 0-100-mm region. A variety of denitrifiers such as Pseudoxanthomona, Acidovorax, and Simplicispira were enriched, which probably were responsible for both NO3--N and Cr(VI) removal. Our findings advance the understanding of simultaneous nitrate and chromium removal in pyrite-involved mixotrophic systems and facilitate the new strategy development for nitrate and chromium remediation.

Shotgun metagenomes from productive lakes in an urban region of Sweden

Urban lakes provide multiple benefits to society while influencing life quality. Moreover, lakes and their microbiomes are sentinels of anthropogenic impact and can be used for natural resource management and planning. Here, we release original metagenomic data from several well-characterized and anthropogenically impacted eutrophic lakes in the vicinity of Stockholm (Sweden). Our goal was to collect representative microbial community samples and use shotgun sequencing to provide a broad view on microbial diversity of productive urban lakes. Our dataset has an emphasis on Lake Mälaren as a major drinking water reservoir under anthropogenic impact. This dataset includes short-read sequence data and metagenome assemblies from each of 17 samples collected from eutrophic lakes near the greater Stockholm area. We used genome-resolved metagenomics and obtained 2378 metagenome assembled genomes that de-replicated into 514 species representative genomes. This dataset adds new datapoints to previously sequenced lakes and it includes the first sequenced set of metagenomes from Lake Mälaren. Our dataset serves as a baseline for future monitoring of drinking water reservoirs and urban lakes.

Regenerative endodontic procedures in immature permanent teeth with pulp necrosis: the impact of microbiology on the clinical and radiographic outcome

AimsThe current study aimed to determine how the regenerative endodontic procedures (REP) disinfection strategy influences overall bacterial load and REP outcomes. Different bacterial species in the teeth were also examined.MethodsA previously reported non-randomized controlled clinical research on REP ± leukocyte and platelet-rich fibrin gathered microbial samples from 14 of 29 patients during REP (LPRF). Four microbiological samples were obtained in two treatment sessions. S1 and S2 were taken before and after the first irrigation with 1.5% NaOCl and saline. Samples S3 and S4 were obtained before and after rinsing with 17% EDTA in the second treatment session. Microbial samples were identified using quantitative polymerase chain reaction with species-specific primers.ResultsThe total bacterial load recovered from patients showed a significant (p &amp;lt; 0.05) decrease after the first treatment and was maintained throughout the second treatment. Fusobacterium nucleatum, Treponema denticola, and Enterococcus faecalis were the most prevalent species in root canals, detected in all analyzed cases (100%), followed by Prevotella intermedia and Tannerella forsythia, both in six of 14 (42.9%) cases. The presence of these abundant species was significantly reduced after S1. Parvimonas micra was present in four of 14 (28.6%) cases and Actinomyces naeslundii in two of 14 (14.3%) cases. Filifactor alocis, Porphyromonas endodontalis, and Porphyromonas gingivalis were each detected in only one of 14 (7.1%) cases. No statistical correlation could be made between bacterial species and clinical or radiographic outcomes due to the small sample size. In the LPRF group, two cases required retreatment due to early post-treatment flare-up, and two other cases presented radiographically a persistent apical periodontitis 3 years after treatment. In the control group, all analyzed cases were clinically asymptomatic after treatment, and radiographically the final periapical index score at the last recall revealed healthy periapices.ConclusionThe REP disinfection protocol of the present study seems to be satisfactorily effective in reducing the total bacterial load, omitting clinical symptoms, and inducing periapical bone healing in immature permanent teeth with pulp necrosis. LPRF seems to prevent these outcomes from being achieved and should consequently not be recommended in REP.

In-vitro antimicrobial efficacy of heterostructured ZnO/ZnWO4 nanocomposites for dental caries

ZnO/ZnWO4 nanocomposites were synthesized in a one-pot green technique utilizing Lycopersicon esculentum extract and were confirmed by PXRD, SEM, EDX, and thermogravimetric analysis. The antimicrobial efficacy of nano-sized ZnO/ZnWO4 against salivary microbial samples was studied using the Well diffusion and time-course analysis methods. The results disclosed that nanocomposite has greater inhibition of the growth of salivary microbes. Further analysis proved that it controls Lactobacillus highly and Streptococcus mutans poorly. This ZnO/ZnWO4 nanocomposites exhibits up to 69% higher inhibition than that of ZnO nano compound and 70% higher activity than commercial ZnO at 130 mg/mL concentration. Thus ZnO/ZnWO4 nanocomposites can be promising candidates to replace the ZnO powder which is currently used for endodontic applications.

The Validity of the Poisson Distribution to Analyze Microbial Colony Counts on Agar Plates for Food Samples

Microbial colony counts of food samples in microbiological examinations are one of the most important items. The probability distributions for the colony counts per agar plate at the dilution of counting had not been intensively studied so far. Recently we analyzed the colony counts of food samples with several probability distributions using the Pearson's chi-square value by the "traditional" statistics as the index of fit [Fujikawa and Tsubaki, Food Hyg.Saf.Sc., 60, 88-95 (2019)]. As a result, the selected probability distributions depended on the samples. In this study we newly selected a probability distribution, namely a statistical model, suitable for the above data with the method of maximum likelihood from the probabilistic point of view. The Akaike's Information Criterion (AIC) was used as the index of fit. Consequently, the Poisson model were better than the negative binomial model for all of four food samples. The Poisson model was also better than the binomial for three of four microbial culture samples. With Baysian Information Criterion (BIC), the Poisson model was also better than these two models for all the samples. These results suggested that the Poisson distribution would be the best model to estimate the colony counts of food samples. The present study would be the first report on the statistical model selection for the colony counts of food samples with AIC and BIC.

Characteristics of Gastric Mucosa-Associated Microbiota in Patients with Early Gastric Cancer After Successful Helicobacter pylori Eradication.

Helicobacter pylori (H. pylori) is widely recognized as a definite carcinogen in gastric cancer (GC). Although H. pylori eradication reduces the risk of GC, GC recurrence has been detected even after successful H. pylori eradication. Recently, the analysis of gut microbiota was reported. This study aimed to evaluate the correlation between gastric mucosa-associated microbiota (G-MAM) and early gastric cancer (EGC) after successful H. pylori eradication. In this pilot study, G-MAM were collected during the esophagogastroduodenoscopy of 17 patients, receiving H. pylori eradication therapy at least 5years ago. The patients were divided into those with EGC (the EGC group, 8 patients) and those without EGC (the NGC group, 9 patients). Microbial samples in the greater curvature of the pyloric site were obtained using an endoscopic cytology brush, and the G-MAM profiles of each sample were analyzed using 16S rRNA V3-V4 gene sequencing. Between the two groups, there was no significant difference in the median age, sex, median period after successful eradication of H. pylori, the α diversity, and the average abundance at the phylum level. At the genus level, the average abundance of Unclassified Oxalobacteraceae, Capnocytophaga, and Haemophilus was significantly lower in the EGC group than in the NGC group (0.89 vs. 0.14%, P < 0.01, 0.28 vs. 0.00%, P < 0.01 and 5.84 vs. 2.16%, P = 0.034, respectively). We demonstrated alternations in the profiles of G-MAM between the two groups. Our results suggest that G-MAM may influence carcinogenesis after successful H. pylori eradication.

Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Microarray Polymer Profiling (MAPP) for High-Throughput Glycan Analysis.

Microarray polymer profiling (MAPP) is a robust and reproducible approach to systematically determine the composition and relative abundance of glycans and glycoconjugates within a variety of biological samples, including plant and algal tissues, food materials, and human, animal, and microbial samples. Microarray technology underpins the efficacy of this method by providing a miniaturized, high-throughput screening platform, allowing thousands of interactions between glycans and highly specific glycan-directed molecular probes to be characterized concomitantly, using only small amounts of analytes. Constituent glycans are chemically and enzymatically fractionated, before being sequentially extracted from the sample and directly immobilized onto nitrocellulose membranes. The glycan composition is determined by the attachment of specific glycan-recognizing molecular probes to the extorted and printed molecules. MAPP is complementary to conventional glycan analysis techniques, such as monosaccharide and linkage analysis and mass spectrometry. However, glycan-recognizing molecular probes provide insight into the structural configurations of glycans, which can aid in elucidating biological interactions and functional roles.

Incidence, microbiological and immunological characteristics of ventilator-associated pneumonia assessed by bronchoalveolar lavage and endotracheal aspirate in a prospective cohort of COVID-19 patients: CoV-AP study

BackgroundNo univocal recommendation exists for microbiological diagnosis of ventilator-associated pneumonia (VAP). Sampling of either proximal or distal respiratory tract likely impacts on the broad range of VAP incidence between cohorts. Immune biomarkers to rule-in/rule-out VAP diagnosis, although promising, have not yet been validated. COVID-19-induced ARDS made VAP recognition even more challenging, often leading to overdiagnosis and overtreatment. We evaluated the impact of different respiratory samples and laboratory techniques on VAP incidence and microbiological findings in COVID-19 patients.MethodsProspective single-centre cohort study conducted among COVID-19 mechanically ventilated patients in Policlinico Hospital (Milan, Italy) from January 2021 to May 2022. Microbiological confirmation of suspected VAP (sVAP) was based on concomitant endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL). Conventional and fast microbiology (FILMARRAY® Pneumonia Panel plus, BALFAPPP) as well as immunological markers (immune cells and inflammatory cytokines) was analysed.ResultsSeventy-nine patients were included. Exposure to antibiotics and steroid therapy before ICU admission occurred in 51/79 (64.6%) and 60/79 (65.9%) patients, respectively. Median duration of MV at VAP suspicion was 6 (5–9) days. Incidence rate of microbiologically confirmed VAP was 33.1 (95% CI 22.1–44.0) and 20.1 (95% CI 12.5–27.7) according to ETA and BAL, respectively. Concordance between ETA and BAL was observed in 35/49 (71.4%) cases, concordance between BALFAPPP and BAL in 39/49 (79.6%) cases. With BAL as reference standard, ETA showed 88.9% (95% CI 70.8–97.7) sensitivity and 50.0% (95% CI 28.2–71.8) specificity (Cohen’s Kappa 0.40, 95% CI 0.16–0.65). BALFAPPP showed 95.0% (95% CI 75.1–99.9) sensitivity and 69% (95% CI 49.2–84.7) specificity (Cohen’s Kappa 0.60, 95% CI 0.39–0.81). BAL IL-1β differed significantly between VAP (135 (IQR 11–450) pg/ml) and no-VAP (10 (IQR 2.9–105) pg/ml) patients (P = 0.03).ConclusionsIn COVID-19 ICU patients, differences in microbial sampling at VAP suspicion could lead to high variability in VAP incidence and microbiological findings. Concordance between ETA and BAL was mainly limited by over 20% of ETA positive and BAL negative samples, while BALFAPPP showed high sensitivity but limited specificity when evaluating in-panel targets only. These factors should be considered when comparing results of cohorts with different sampling. BAL IL-1β showed potential in discriminating microbiologically confirmed VAP.Clinical Trial registration: NCT04766983, registered on February 23, 2021.