Summary
Metagenomics provide unprecedented insights into the genetic diversity of uncultivated bacteria inhabiting natural environments. Recent surveys have uncovered a major radiation of candidate ...phyla encompassing the Patescibacteria superphylum. Patescibacteria have small genomes and a presumed symbiotic or parasitic lifestyle, but the difficulty in culturing representative members constrains the characterization of behavioural and adaptive traits. Here we combine in silico and in vitro approaches to characterize the SOS transcriptional response to DNA damage in the Patescibacteria superphylum. Leveraging comparative genomics methods, we identify and experimentally define a novel binding motif for the SOS transcriptional repressor LexA, and we use this motif to characterize the conserved elements of the SOS regulatory network in Patescibacteria. The Patescibacteria LexA‐binding motif has unusual direct‐repeat structure, and comparative analyses reveal sequence and structural similarities with the distant Acidobacteria LexA protein. Our results reveal a shared core SOS network, complemented by varying degrees of LexA regulation of other core SOS functions. This work illustrates how the combination of computational and experimental methods can leverage metagenomic data to characterize transcriptional responses in uncultivated bacteria. The report of an operational SOS response in presumed symbiotic and parasitic bacteria hints at an intermediate step in the process of genome reduction.
Sulfonamides are synthetic chemotherapeutic agents that work as competitive inhibitors of the di-hydro-pteroate synthase (DHPS) enzyme, encoded by the
gene. Resistance to sulfonamides is widespread ...in the clinical setting and predominantly mediated by plasmid- and integron-borne
genes encoding mutant DHPS enzymes that do not bind sulfonamides. In spite of their clinical importance, the genetic origin of
genes remains unknown. Here we analyze
genes and their genetic neighborhoods to uncover
signature elements that enable the elucidation of their genetic origin. We identify a protein sequence Sul motif associated with
-encoded proteins, as well as consistent association of a phosphoglucosamine mutase gene (
) with the
gene. We identify chromosomal
genes bearing these genetic markers in two bacterial families: the
and the
. Bayesian phylogenetic inference of FolP/Sul and GlmM protein sequences clearly establishes that
and
genes originated as a mobilization of
genes present in, respectively, the
and the
, and indicate that the
gene was transferred from the
. Analysis of %GC content in
/
gene sequences supports the phylogenetic inference results and indicates that the emergence of the Sul motif in chromosomally encoded FolP proteins is ancient and considerably predates the clinical introduction of sulfonamides.
assays reveal that both the
and the
, but not other related chromosomally encoded FolP proteins confer resistance in a sulfonamide-sensitive
background, indicating that the Sul motif is associated with sulfonamide resistance. Given the absence of any known natural sulfonamides targeting DHPS, these results provide a novel perspective on the emergence of resistance to synthetic chemotherapeutic agents, whereby preexisting resistant variants in the vast bacterial pangenome may be rapidly selected for and disseminated upon the clinical introduction of novel chemotherapeuticals.
Abstract
Objective
To apply lean thinking in triage acuity level-3 patients in order to improve emergency department (ED) throughtput and waiting time.
Design
A prospective interventional study.
...Setting
An ED of a tertiary care hospital.
Participants
Triage acuity level-3 patients.
Intervention(s)
To apply lean techniques such as value stream mapping, workplace organization, reduction of wastes and standardization by the frontline staff.
Main Outcome Measure(s)
Two periods were compared: (i) pre-lean: April–September, 2015; and (ii) post-lean: April–September, 2016. Variables included: median process time (time from beginning of nurse preparation to the end of nurse finalization after doctor disposition) of both discharged and transferred to observation patients; median length of stay; median waiting time; left without being seen, 72-h revisit and mortality rates, and daily number of visits. There was no additional staff or bed after lean implementation.
Results
Despite an increment in the daily number of visits (+8.3%, P < 0.001), significant reductions in process time of discharged (182 vs 160 min, P < 0.001) and transferred to observation (186 vs 176 min, P < 0.001) patients, in length of stay (389 vs 329 min, P < 0.001), and in waiting time (71 vs 48 min, P < 0.001) were achieved after lean implementation. No significant differences were registered in left without being seen rate (5.23% vs 4.95%), 72-h revisit rate (3.41% vs 3.93%), and mortality rate (0.23% vs 0.15%).
Conclusion
Lean thinking is a methodology that can improve triage acuity level-3 patient flow in the ED, resulting in better throughput along with reduced waiting time.
Bacterial EVs have been related to inter-kingdom communication between probiotic/pathogenic bacteria and their hosts. Our aim was to investigate the transcytosis process of B. subtilis EVs using an ...in vitro intestinal epithelial cell model. In this study, using Confocal Laser Scanning Microscopy, we report that uptake and internalization of CFSE-labeled B. subtilis EVs (115 nm ± 27 nm) by Caco-2 cells are time-dependent. To study the transcytosis process we used a transwell system and EVs were quantified in the lower chamber by Fluorescence and Nanoparticle Tracking Analysis measurements. Intact EVs are transported across a polarized cell monolayer at 60-120 min and increased after 240 min with an estimated average uptake efficiency of 30% and this process is dose-dependent. EVs movement into intestinal epithelial cells was mainly through Z axis and scarcely on X and Y axis. This work demonstrates that EVs could be transported across the gastrointestinal epithelium. We speculate this mechanism could be the first step allowing EVs to reach the bloodstream for further delivery up to extraintestinal tissues and organs. The expression and further encapsulation of bioactive molecules into natural nanoparticles produced by probiotic bacteria could have practical implications in food, nutraceuticals and clinical therapies.
Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move ...by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND resistance nodulation division component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.
Comparative genomics methods enable the reconstruction of bacterial regulatory networks using available experimental data. In spite of their potential for accelerating research into the composition ...and evolution of bacterial regulons, few comparative genomics suites have been developed for the automated analysis of these regulatory systems. Available solutions typically rely on precomputed databases for operon and ortholog predictions, limiting the scope of analyses to processed complete genomes, and several key issues such as the transfer of experimental information or the integration of regulatory information in a probabilistic setting remain largely unaddressed.
Here we introduce CGB, a flexible platform for comparative genomics of prokaryotic regulons. CGB has few external dependencies and enables fully customized analyses of newly available genome data. The platform automates the merging of experimental information and uses a gene-centered, Bayesian framework to generate and integrate easily interpretable results. We demonstrate its flexibility and power by analyzing the evolution of type III secretion system regulation in pathogenic Proteobacteria and by characterizing the SOS regulon of a new bacterial phylum, the Balneolaeota.
Our results demonstrate the applicability of the CGB pipeline in multiple settings. CGB's ability to automatically integrate experimental information from multiple sources and use complete and draft genomic data, coupled with its non-reliance on precomputed databases and its easily interpretable display of gene-centered posterior probabilities of regulation provide users with an unprecedented level of flexibility in launching comparative genomics analyses of prokaryotic transcriptional regulatory networks. The analyses of type III secretion and SOS response regulatory networks illustrate instances of convergent and divergent evolution of these regulatory systems, showcasing the power of formal ancestral state reconstruction at inferring the evolutionary history of regulatory networks.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The emergence of pathogenic strains resistant to multiple antimicrobials is a pressing problem in modern healthcare. Antimicrobial resistance is mediated primarily by dissemination of resistance ...determinants via horizontal gene transfer. The dissemination of some resistance genes has been well documented, but few studies have analyzed the patterns underpinning the dissemination of antimicrobial resistance genes. Analyzing the %GC content of plasmid-borne antimicrobial resistance genes relative to their host genome %GC content provides a means to efficiently detect and quantify dissemination of antimicrobial resistance genes. In this work we automate %GC content analysis to perform a comprehensive analysis of known antimicrobial resistance genes in publicly available plasmid sequences. We find that the degree to which antimicrobial resistance genes are disseminated depends primarily on the resistance mechanism. Our analysis identifies conjugative plasmids as primary dissemination vectors and indicates that most broadly disseminated genes have spread from single genomic backgrounds. We show that resistance dissemination profiles vary greatly among antimicrobials, oftentimes reflecting stewardship measures. Our findings establish %GC content analysis as a powerful, intuitive and scalable method to monitor the dissemination of resistance determinants using publicly available sequence data.
Conducted emissions (CE) for three-phase systems are becoming an increasing concern due to the recent exponential growth of three-phase applications, especially linked to the automotive sector. The ...problem arises because electromagnetic compatibility (EMC) standards only define the methodology to measure the CE generated by the equipment under test (EUT), and they do not provide sufficient information to design a power line filter (PLF) in case of non-compliance. Hence, the design of an optimal PLF is a very difficult task for engineers. The unclear methodology to be followed, unknown load impedances, inadequate equipment, and lack of knowledge of the modal noise are all different reasons that contribute to increasing the PLF design complexity. Common mode (CM) and differential mode (DM) decomposition and PLF design techniques for single-phase EUTs are well discussed and studied in the literature, but the same cannot be stated when it comes to three-phase PLF design. The objective of this paper is to clarify how modal noises behave in a three-phase system and to propose a clear methodology which can be followed to design an optimal three-phase PLF. Additionally, this paper analyses and discusses the modal noises’ intrinsic behavior and provides an understanding of how the PLF components behave when subjected to either a CM or DM noise. Finally, a methodology to design a three-phase PLF, based on accurate insertion loss (IL) estimations and S-parameter measurements, is used to determine the optimal PLF. This approach is tested and validated.
Introduction
The emergence of resistance and interference mechanisms to phage infection can hinder the success of bacteriophage-based applications, but the significance of these mechanisms in phage ...therapy has not been determined. This work studies the emergence of
Salmonella
isolates with reduced susceptibility to a cocktail of three phages under three scenarios: i)
Salmonella
cultures (LAB), ii) biocontrol of cooked ham slices as a model of food safety (FOOD), and iii) oral phage therapy in broilers (PT).
Methods
S
. Typhimurium ATCC 14028 RifR variants with reduced phage susceptibility were isolated from the three scenarios and conventional and molecular microbiology techniques were applied to study them.
Results and discussion
In LAB, 92% of
Salmonella
isolates lost susceptibility to all three phages 24 h after phage infection. This percentage was lower in FOOD, with 4.3% of isolates not susceptible to at least two of the three phages after seven days at 4°C following phage treatment. In PT, 9.7% and 3.3 % of isolates from untreated and treated broilers, respectively, displayed some mechanism of interference with the life cycle of some of the phages. In LAB and FOOD scenarios, resistant variants carrying mutations in
rfc
and
rfaJ
genes involved in lipopolysaccharide synthesis (phage receptor) were identified. However, in PT, the significant decrease of EOP, ECOI, and burst size observed in isolates was prompted by lateral gene transfer of large IncI1 plasmids, which may encode phage defense mechanisms. These data indicate that the acquisition of specific conjugative plasmids has a stronger impact than mutagenesis on the emergence of reduced phage-susceptibility bacteria in certain environments. In spite of this, neither mechanism seems to significantly impair the success of
Salmonella
biocontrol and oral phage therapy.
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