To accelerate differentiation between Staphylococcus aureus and Coagulase Negative Staphylococci (CNS), this study aimed to compare six different DNA extraction methods from 2 commonly used blood culture materials, i.e. BACTEC and Bact/ALERT. Furthermore, we analyzed the effect of reduced blood culture times for detection of Staphylococci directly from blood culture material. A real-time PCR duplex assay was used to compare 6 different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with MRSA. Bacterial DNA was isolated with: automated extractor EasyMAG (3 protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus, and a combination between MolYsis Plus and the EasyMAG. The most optimal isolation method was used to evaluate reduced bacterial culture times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the EasyMAG resulted in the most sensitive detection of S.aureus, with a detection limit of 10 CFU/ml, in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S.aureus load of 1 CFU/ml blood can be detected after 5 hours of culture in Bact/ALERT3D by combining the sensitive isolation method and the tuf LightCycler assay.
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Five methods were compared to determine the best technique for accurate identification of coagulase-negative staphylococci (CoNS) (n=142 strains). MALDI-TOF MS showed the best results for rapid and accurate CoNS differentiation (correct identity in 99.3%). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing.
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IL22 is an important cytokine involved in the intestinal defense mechanisms against microbiome. By using ileum-derived organoids, we show that the expression of anti-microbial peptides (AMPs) and anti-viral peptides (AVPs) can be induced by IL22. In addition, we identified a bacterial and a viral route, both leading to IL22 production by T cells, but via different pathways. Bacterial products, such as LPS, induce enterocyte-secreted SAA1, which triggers the secretion of IL6 in fibroblasts, and subsequently IL22 in T cells. This IL22 induction can then be enhanced by macrophage-derived TNFα in two ways: by enhancing the responsiveness of T cells to IL6 and by increasing the expression of IL6 by fibroblasts. Viral infections of intestinal cells induce IFNβ1 and subsequently IL7. IFNβ1 can induce the expression of IL6 in fibroblasts and the combined activity of IL6 and IL7 can then induce IL22 expression in T cells. We also show that IL22 reduces the expression of viral entry receptors (e.g. ACE2, TMPRSS2, DPP4, CD46 and TNFRSF14), increases the expression of anti-viral proteins (e.g. RSAD2, AOS, ISG20 and Mx1) and, consequently, reduces the viral infection of neighboring cells. Overall, our data indicates that IL22 contributes to the innate responses against both bacteria and viruses.
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Matrix-assisted laser desorption/ionisation time of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria from agar media. Direct identification from positive blood cultures should decrease the time to obtaining the result. In this study, three different processing methods for the rapid direct identification of bacteria from positive blood culture bottles were compared. In total, 101 positive aerobe BacT/ALERT bottles were included in this study. Aliquots from all bottles were used for three bacterial processing methods, i.e. the commercially available Bruker's MALDI Sepsityper kit, the commercially available Molzym's MolYsis Basic5 kit and a centrifugation/washing method. In addition, the best method was used to evaluate the possibility of MALDI application after a reduced incubation time of 7 h of Staphylococcus aureus- and Escherichia coli-spiked (1,000, 100 and 10 colony-forming units [CFU]) aerobe BacT/ALERT blood cultures. Sixty-six (65%), 51 (50.5%) and 79 (78%) bottles were identified correctly at the species level when the centrifugation/washing method, MolYsis Basic 5 and Sepsityper were used, respectively. Incorrect identification was obtained in 35 (35%), 50 (49.5%) and 22 (22%) bottles, respectively. Gram-positive cocci were correctly identified in 33/52 (64%) of the cases. However, Gram-negative rods showed a correct identification in 45/47 (96%) of all bottles when the Sepsityper kit was used. Seven hours of pre-incubation of S. aureus- and E. coli-spiked aerobe BacT/ALERT blood cultures never resulted in reliable identification with MALDI-TOF MS. Sepsityper is superior for the direct identification of microorganisms from aerobe BacT/ALERT bottles. Gram-negative pathogens show better results compared to Gram-positive bacteria. Reduced incubation followed by MALDI-TOF MS did not result in faster reliable identification.
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Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome‐wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site‐directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101.
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Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.
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Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman’s menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.
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tIn this study we aimed to identify genes that are responsive to pertussis toxin (PTx) and might eventu-ally be used as biological markers in a testing strategy to detect residual PTx in vaccines. By microarrayanalysis we screened six human cell types (bronchial epithelial cell line BEAS-2B, fetal lung fibroblastcell line MRC-5, primary cardiac microvascular endothelial cells, primary pulmonary artery smooth mus-cle cells, hybrid cell line EA.Hy926 of umbilical vein endothelial cells and epithelial cell line A549 andimmature monocyte-derived dendritic cells) for differential gene expression induced by PTx. Imma-ture monocyte-derived dendritic cells (iMoDCs) were the only cells in which PTx induced significantdifferential expression of genes. Results were confirmed using different donors and further extendedby showing specificity for PTx in comparison to Escherichia coli lipopolysaccharide (LPS) and Bordetellapertussis lipo-oligosaccharide (LOS). Statistical analysis indicated 6 genes, namely IFNG, IL2, XCL1, CD69,CSF2 and CXCL10, as significantly upregulated by PTx which was also demonstrated at the protein levelfor genes encoding secreted proteins. IL-2 and IFN- gave the strongest response. The minimal PTx con-centrations that induced production of IL-2 and IFN- in iMoDCs were 12.5 and 25 IU/ml, respectively.High concentrations of LPS slightly induced IFN- but not IL-2, while LOS and detoxified pertussis toxindid not induce production of either cytokine. In conclusion, using microarray analysis we evaluated sixhuman cell lines/types for their responsiveness to PTx and found 6 PTx-responsive genes in iMoDCs ofwhich IL2 is the most promising candidate to be used as a biomarker for the detection of residual PTx.
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The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00–2.49 log CFU/g of manured soil, ranging between 10–5 and 10–4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.
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