Iskanje po repozitoriju

A+ | A- | | SLO | ENG

Povzetek: This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics. To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire. Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens.
Ključne besede: nucleic acid tests, infectious diseases, virus detection, bacteria detection, isothermal nucleic acid amplification, status report, questionnaire, NAT, PCR
Objavljeno v DiRROS: 26.07.2024; Ogledov: 12; Prenosov: 9
Celotno besedilo (5,32 MB)
Gradivo ima več datotek! Več...

Povzetek: Background The Dickeya genus is part of the Pectobacteriaceae family that is included in the newly described enterobacterales order. It comprises a group of aggressive soft rot pathogens with wide geographic distribution and host range. Among them, the new Dickeya fangzhongdai species groups causative agents of maceration-associated diseases that impact a wide variety of crops and ornamentals. It affects mainly monocot plants, but D. fangzhongdai strains have also been isolated from pear trees and water sources. Here, we analysed which genetic novelty exists in this new species, what are the D. fangzhongdai-specific traits and what is the intra-specific diversity. Results The genomes of eight D. fangzhongdai strains isolated from diverse environments were compared to 31 genomes of strains belonging to other Dickeya species. The D. fangzhongdai core genome regroups approximately 3500 common genes, including most genes that encode virulence factors and regulators characterised in the D. dadantii 3937 model strain. Only 38 genes are present in D. fangzhongdai and absent in all other Dickeyas. One of them encodes a pectate lyase of the PL10 family of polysaccharide lyases that is found only in a few bacteria from the plant environment, soil or human gut. Other D. fangzhongdai-specific genes with a known or predicted function are involved in regulation or metabolism. The intra-species diversity analysis revealed that seven of the studied D. fangzhongdai strains were grouped into two distinct clades. Each clade possesses a pool of 100–150 genes that are shared by the clade members, but absent from the other D. fangzhongdai strains and several of these genes are clustered into genomic regions. At the strain level, diversity resides mainly in the arsenal of T5SS- and T6SS-related toxin-antitoxin systems and in secondary metabolite biogenesis pathways. Conclusion This study identified the genome-specific traits of the new D. fangzhongdai species and highlighted the intra-species diversity of this species. This diversity encompasses secondary metabolites biosynthetic pathways and toxins or the repertoire of genes of extrachromosomal origin. We however didn’t find any relationship between gene content and phenotypic differences or sharing of environmental habitats. Background Soft rot Pectobacteriaceae are Enterobacterales responsible for considerable economic losses in several important crops and ornamental plants [1,2,3]. Their virulence is mainly due to the production and secretion of a battery of plant cell wall degrading enzymes (PCWDEs) that cause maceration of the plant tissue; however, several other virulence factors have also been characterized [2, 4]. These bacteria often exhibit a very broad host range, and recent outbreaks in potato, for example, resulted from the action of a cohort of bacteria belonging to different Pectobacteriaceae species in a complex population dynamics history [5]. The Pectobacteriaceae family includes two genera comprising soft rot bacteria, Pectobacterium and Dickeya. The Dickeya genus was formed in 2005 by the reclassification of former Erwinia chrysanthemi into six species [6]. It has recently undergone multiple phylogenetic changes, including the addition of three new species, Dickeya solani [7], Dickeya aquatica [8] and, more recently, Dickeya fangzhongdai [9]. The description of this last new species was based on three isolates from pear trees in China with bleeding canker necrosis [9], but it was extended by a large number of strains isolated from monocot plants from Japan [10, 11]. D. fangzhongdai strains were associated with soft rot symptoms of many ornamental and economically important staple food plants [10, 12, 13], thereby highlighting the broad host range of the species. While there is little information regarding associated economic damages and the extent of its occurrence in different host plants outside of Asia, Alič et al. [14] recently identified D. fangzhongdai as the causative agent of soft rot of orchids in commercial production in Europe, starting with material from Asia [11]. Moreover, as previously reported, bacteriophages of different families, and active against D. fangzhongdai, were isolated from a wastewater treatment plant not associated to the orchid production site. This would suggest that D. fangzhongdai bacteria may be more widespread in nature than could currently be concluded on the basis of symptoms in plants. Its occurrence in water would suggest that it may potentially have a wider ecological niche than genomically close Dickeya spp., that is, Dickeya dadantii, Dickeya dianthicola, and D. solani. Previous experience with D. solani has shown that novel species or isolates can lead to clonal spread and high losses in affected host plants [15]. Together with repeated introductions of D. fangzhongdai, the co-occurrence of genetically and phenotypically diverse strains on the same plants (e.g., B16 and S1 on orchids, as reported by Alič et al. [11]) increases the probability of the development of recombined strains with novel pathogenic potential and may present a risk to agriculturally important plants. Their aggressiveness, high maceration potential on various plant tissues, and persistence in potato plants further exacerbate the risk for agriculture. Therefore, in this paper, we analysed the genomic characteristics of the D. fangzhongdai species, compared it to the other Dickeya species and determine the inter- and intra- species diversity. The study addressed the question whether the presence of the isolates in a specific environment is associated to a specific set of genes (water vs plant symptoms, monocots vs dicots, different geographical origin). We also analysed the virulence gene arsenal, in order to evaluate the virulence potential of this species. Methods Dickeya strain selection All D. fangzhongdai genomes publicly available in the NCBI database were included in this study. These genomes were compared to five D. solani, four D. dadantii, five D. dianthicola, five D. chrysanthemi, seven D. zeae, one D. aquatica, two D. paradisiaca and two unassigned Dickeya genomes extracted from the NCBI database. Information on the provenance and genomic data of the D. fangzhongdai strains used in this study are summarized in Table 1. The accession numbers and phylogenetic position of the other Dickeya strains used for the SiLix analyses are presented in Additional file 1: Figure S1.
Ključne besede: T5SS, T6SS, NRPS/PKS, zeamine, oocydin A, plant-bacteria interactions, plasmid, Dickeya fangzhongdai
Objavljeno v DiRROS: 23.07.2024; Ogledov: 39; Prenosov: 7
Celotno besedilo (2,95 MB)
Gradivo ima več datotek! Več...

Povzetek: Background Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803. Methods We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target. Results With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
Ključne besede: bacteria, cyanobacteria, synthetic biology, proteolytic cleavage, cystatin
Objavljeno v DiRROS: 19.07.2024; Ogledov: 44; Prenosov: 21
Celotno besedilo (3,66 MB)
Gradivo ima več datotek! Več...

Povzetek: Background: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. Results: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. Conclusions: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.
Ključne besede: whole-genome assembly, non-axenic culture, plasmid, marine bacteria, marine biology
Objavljeno v DiRROS: 28.03.2024; Ogledov: 343; Prenosov: 174
Celotno besedilo (5,87 MB)
Gradivo ima več datotek! Več...

Povzetek: Objectives: The aim was to quantify the effects of selective digestive tract decontamination (SDD) consisting of a mouth paste and gastro-enteral suspension, selective oropharyngeal decontamination with a mouth paste (SOD) and 1-2% chlorhexidine (CHX) mouthwash on eradication and acquisition of carriage of third-generation cephalosporin-resistant Enterobacterales (3GCR-E) and carbapenem-resistant Gram-negative bacteria (CR-GNB) in Intensive Care Unit (ICU) patients. Methods: This was a nested cohort study within a cluster-randomized cross-over trial in six European countries and 13 ICUs with 8665 patients. Eradication and acquisition during ICU stay of 3GCR-E and CRGNB were investigated separately in the rectum and respiratory tract for the three interventions and compared with standard care (SC) using Cox-regression competing events analyses. Results: Adjusted cause specific hazard ratios (CSHR) for eradication of rectal carriage for SDD were 1.76 (95% CI 1.31-2.36) for 3GCR-E and 3.17 (95% CI 1.60-6.29) for CR-GNB compared with SC. For the respiratory tract, adjusted CSHR for eradication of 3GCR-E were 1.47 (0.98-2.20) for SDD and 1.38 (0.92-2.06) for SOD compared with SC, and for eradication of CR-GNB these were 0.77 (0.41-1.45) for SDD and 0.81 (0.44-1.51) for SOD, compared with SC. Adjusted CSHRs for acquisition of rectal carriage during SDD (compared with SC) were 0.51 (0.40-0.64) for 3GCR-E and of 0.56 (0.40-0.78) for CR-GNB. Adjusted CSHRs for acquiring respiratory tract carriage with 3GCR-E compared with SC were 0.38 (0.28-0.50) for SDD and 0.55 (0.42-0.71) for SOD, and for CR-GNB 0.46 (0.33-0.64) during SDD and 0.60 (0.44-0.81) during SOD, respectively. SOD was not associated with eradication or acquisition of 3GCR-E and CR-GNB in the rectum. Conclusions: Among mechanically ventilated ICU patients, SDD was associated with more eradication and less acquisition of 3GCR-E and CR-GNB in the rectum than SC. SDD and SOD were associated with less acquisition of both 3GCR-E and CR-GNB than SC in the respiratory tract.
Ključne besede: intensive care units -- analysis -- epidemiology, bacterial drug resistance, anti-infective agents -- therapeutic use decontamination, beta-lactamases, Gram-negative bacteria, gastrointestinal tract -- microbiology -- drug therapy, cohort studies, colonization, ESBL, digestive tract
Objavljeno v DiRROS: 27.05.2022; Ogledov: 659; Prenosov: 252
Povezava na datoteko