Emerging Campylobacter and Arcobacter spp. have been increasingly isolated from human clinical samples, food, veterinary samples and the environment. Unambiguous species identification of such ...organisms is of obvious importance in epidemiological studies, but is also necessary to accurately assess their host range and determine their prevalence in the food chain and in the environment. Species identification methods for the Campylobacteraceae have been described; however, some with high resolving power are limited to a small number of taxa, while other broader‐range methods cannot distinguish between closely related species. We present in this study a novel species identification method, based on amplification and sequencing of a portion of the atpA gene. This method, which uses a single primer pair, was able to amplify and accurately identify all current taxa within Campylobacter and Arcobacter as well as several members of the Helicobacteraceae, although unambiguous identification of the Camp. fetus subspecies could not be achieved. In addition, five putative novel Campylobacter taxa were recognized, making this new species identification method valuable in the characterization of novel epsilonproteobacteria. Thus, a single‐locus method that can accurately identify multiple epsilonproteobacterial species will prove important in the characterization of emerging organisms and those associated with illness. SIGNIFICANCE AND IMPACT OF THE STUDY: The atpA‐based species identification method described here uses a single primer pair to amplify DNA from all current validly‐described Campylobacter and Arcobacter taxa, as well as multiple members of the Helicobacteraceae. This method unambiguously identified all taxa tested, although it could not discriminate the subspecies of Camp. fetus. Furthermore, five putative novel Campylobacter taxa were observed following testing of environmental campylobacters with this method. The scope and resolution of this method make it an important addition to studies of epsilonproteobacterial epidemiology and evolution.
The 32-bit Windows application START is implemented using Visual Basic and C++ and performs analyses to aid in the investigation of bacterial population structure using multilocus sequence data. ...These analyses include data summary, lineage assignment, and tests for recombination and selection. Availability: START is available at http://outbreak.ceid.ox.ac.uk/software.htm. Contact: keith.jolley@ceid.ox.ac.uk * To whom correspondence should be addressed.
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•Recombinant IFITM3 binds to cholesterol in vitro.•Binding maps to the amphipathic helix in the intramembrane domain.•Loss-of-function mutations in IFITM3 disrupt helical structure ...and reduce cholesterol binding.•Cholesterol binding potential in vitro is correlated with membrane insertion depth of the amphipathic helix in silico.
The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.
Xanthomonas citri subsp.
is the cause of bacterial citrus canker, responsible for major economic losses to the citrus industry. X. citri subspecies and pathovars are responsible for diseases in ...soybean, common bean, mango, pomegranate, and cashew.
disease has been tracked using several typing methods, but recent studies using genomic sequencing have been key to understanding the evolutionary relationships within the species, including fundamental differences among
subsp.
pathotypes. Here, we describe a core-genome multilocus sequence typing (cgMLST) scheme for
based on 250 genomes comprising multiple examples of
subsp.
pathotypes A, A*, and A
;
subsp.
;
pv.
, pv.
, pv.
, pv.
, pv.
, and pv.
; and single isolates of
pv.
and pv.
. This data set included genomic sequencing of 100 novel
subsp.
isolates. cgMLST, based on 1,618 core genes across 250 genomes, is implemented at PubMLST (https://pubmlst.org/organisms/xanthomonas-citri/). GrapeTree minimum-spanning tree and Interactive Tree of Life (iTOL) neighbor-joining phylogenies generated from the cgMLST data resolved almost identical groupings of isolates to a core-genome single nucleotide polymorphism (SNP)-based neighbor-joining phylogeny. These resolved identical groupings of
subsp.
pathotypes and
subspecies and pathovars.
cgMLST should prove to be an increasingly valuable resource for the study of this key species of plant-pathogenic bacteria. Users can submit genomic data and associated metadata for comparison with previously characterized isolates at PubMLST to allow the rapid characterization of the local, national, and global epidemiology of these pathogens and examine evolutionary relationships.
Xanthomonas citri is a plant pathogen that causes major economic losses to the citrus industry and sweet orange production in particular. Several subspecies and pathogens are recognized, with host ranges including soybean, common bean, mango, pomegranate, and cashew, among others. Recent genomic studies have shown that host-adapted
subspecies and pathovars and
subsp.
pathotypes form distinct clades. In this study, we describe a core-genome multilocus sequence typing (cgMLST) scheme for this species that can rapidly and robustly discriminate among these ecologically distinct, host-adapted clades. We have established this scheme and associated databases containing genomic sequences and metadata at PubMLST, which users can interrogate with their own genome sequences to determine
subspecies, pathovars, and pathotypes.
cgMLST should prove to be an invaluable tool for the study of the epidemiology and evolution of this major plant pathogen.
Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely phenotypes of aetiological agents, ...enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that many microbiological typing methods have addressed over the last 100 years or so; indeed, providing large volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites have generated an infrastructure that can exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST website to extract clinically useful, strain-characterisation information that can be provided to physicians and public health professionals in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to instigate public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data obtained in the absence of WGS, providing a flexible infrastructure for WGS-based clinical microbiology.
Whole genome sequence (WGS) data are increasingly used to characterise bacterial pathogens. These data provide detailed information on the genotypes and likely phenotypes of aetiological agents, ...enabling the relationships of samples from potential disease outbreaks to be established precisely. However, the generation of increasing quantities of sequence data does not, in itself, resolve the problems that many microbiological typing methods have addressed over the last 100 years or so; indeed, providing large volumes of unstructured data can confuse rather than resolve these issues. Here we review the nascent field of storage of WGS data for clinical application and show how curated sequence-based typing schemes on websites have generated an infrastructure that can exploit WGS for bacterial typing efficiently. We review the tools that have been implemented within the PubMLST website to extract clinically useful, strain-characterisation information that can be provided to physicians and public health professionals in a timely, concise and understandable way. These data can be used to inform medical decisions such as how to treat a patient, whether to instigate public health action, and what action might be appropriate. The information is compatible both with previous sequence-based typing data and also with data obtained in the absence of WGS, providing a flexible infrastructure for WGS-based clinical microbiology.
Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious ...disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.
Objective To estimate medium-term success after a technique for ultrasound-guided sclerotherapy for superficial chronic venous disease. Design A prospective study in a single unit with ultrasound ...surveillance after treatment. Materials Results after 1189 treatment sessions for 807 venous saphenous veins and related tributaries or non-saphenous tributaries in 489 patients. Methods Univariate life table analysis determined primary and secondary success rates. Multivariate Cox regression analysis detected covariates that affected outcome. Results Primary and secondary success rates at 36 months for all veins were 52.4% (95%CI 46–58%) and 76.8% (95%CI 71–82%). Cox regression analysis for primary success for all veins showed significantly worse results for saphenous veins compared to tributaries (HR 3.72 – 95%CI 1.9 to 7.3). Cox regression for all saphenous veins showed independently worse results for patients less than 40 years age (HR 2.16 – 95%CI 1.27–3.66), small compared to great saphenous veins (HR 1.58 – 95%CI 1.11–2.24), veins greater than 6mm diameter compared to smaller veins (HR 2.22 – 95%CI 1.40–3.50), liquid compared to foam sclerotherapy (HR 2.20 – 95%CI 1.28–3.78), lower volumes of sclerosant compared to volumes greater than 12 ml (HR 0.51 – 95%CI 0.33–0.81) and highly diluted compared to concentrated sclerosant (HR 2.05 – 95%CI 1.21–3.46) with worse results using highly diluted or undiluted 3% sclerosant compared to a 1.5% concentration. There were no significant differences for primary success for saphenous veins for date of procedure, sex, side, primary or recurrent varicose veins, or commercial type of sclerosant. Conclusions Ultrasound-guided sclerotherapy gives satisfactory results if it is accepted that treatment may need to be repeated to achieve secondary success. Results provide a basis for further research to explore factors that might affect outcome. Younger patients with larger diameter saphenous veins may warrant alternative forms of treatment, particularly for small saphenous reflux.
Multi-locus sequence typing (MLST) is a method of typing that facilitates the discrimination of microbial isolates by comparing the sequences of housekeeping gene fragments. The mlstdbNet software ...enables the implementation of distributed web-accessible MLST databases that can be linked widely over the Internet.
The software enables multiple isolate databases to query a single profiles database that contains allelic profile and sequence definitions. This separation enables isolate databases to be established by individual laboratories, each customized to the needs of the particular project and with appropriate access restrictions, while maintaining the benefits of a single definitive source of profile and sequence information. Databases are described by an XML file that is parsed by a Perl CGI script. The software offers a large number of ways to query the databases and to further break down and export the results generated. Additional features can be enabled by installing third-party (freely available) tools.
Development of a distributed structure for MLST databases offers scalability and flexibility, allowing participating centres to maintain ownership of their own data, without introducing duplication and data integrity issues.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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