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1.
Front Cell Infect Microbiol ; 11: 634215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34381737

RESUMO

Bloodstream infections (BSIs), the presence of microorganisms in blood, are potentially serious conditions that can quickly develop into sepsis and life-threatening situations. When assessing proper treatment, rapid diagnosis is the key; besides clinical judgement performed by attending physicians, supporting microbiological tests typically are performed, often requiring microbial isolation and culturing steps, which increases the time required for confirming positive cases of BSI. The additional waiting time forces physicians to prescribe broad-spectrum antibiotics and empirically based treatments, before determining the precise cause of the disease. Thus, alternative and more rapid cultivation-independent methods are needed to improve clinical diagnostics, supporting prompt and accurate treatment and reducing the development of antibiotic resistance. In this study, a culture-independent workflow for pathogen detection and identification in blood samples was developed, using peptide biomarkers and applying bottom-up proteomics analyses, i.e., so-called "proteotyping". To demonstrate the feasibility of detection of blood infectious pathogens, using proteotyping, Escherichia coli and Staphylococcus aureus were included in the study, as the most prominent bacterial causes of bacteremia and sepsis, as well as Candida albicans, one of the most prominent causes of fungemia. Model systems including spiked negative blood samples, as well as positive blood cultures, without further culturing steps, were investigated. Furthermore, an experiment designed to determine the incubation time needed for correct identification of the infectious pathogens in blood cultures was performed. The results for the spiked negative blood samples showed that proteotyping was 100- to 1,000-fold more sensitive, in comparison with the MALDI-TOF MS-based approach. Furthermore, in the analyses of ten positive blood cultures each of E. coli and S. aureus, both the MALDI-TOF MS-based and proteotyping approaches were successful in the identification of E. coli, although only proteotyping could identify S. aureus correctly in all samples. Compared with the MALDI-TOF MS-based approaches, shotgun proteotyping demonstrated higher sensitivity and accuracy, and required significantly shorter incubation time before detection and identification of the correct pathogen could be accomplished.


Assuntos
Bacteriemia , Infecções Estafilocócicas , Bacteriemia/diagnóstico , Candida albicans , Escherichia coli , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus
2.
J Cell Physiol ; 236(10): 7256-7265, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33821475

RESUMO

The last two decades have witnessed a tremendous increase in cell biology data. Not least is this true for studies of the dynamic organization of the microfilament and microtubule systems in animal cells where analyses of the molecular components and their interaction patterns have deepened our understanding of these complex force-generating machineries. Previous observations of a molecular cross-talk between the two systems have now led to the realization of the existence of several intricate mechanisms operating to maintain their coordinated cellular organization. In this short review, we relate to this development by discussing new results concerning the function of the actin regulator profilin 1 as a control component of microfilament-microtubule cross-talk.

3.
Sci Adv ; 7(16)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33863724

RESUMO

Several important drug targets, e.g., ion channels and G protein-coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics-sensitive druggability probes in native-state and disease-relevant proteins. By using low-Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

4.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33184056

RESUMO

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.


Assuntos
Actinas/metabolismo , Centrossomo/metabolismo , Melanoma Experimental/metabolismo , Profilinas/metabolismo , Transdução de Sinais/genética , Neoplasias Cutâneas/metabolismo , Animais , Células CACO-2 , Forminas/metabolismo , Técnicas de Inativação de Genes , Humanos , Melanoma Experimental/patologia , Camundongos , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Polimerização , Profilinas/genética , Neoplasias Cutâneas/patologia , Transfecção , Tubulina (Proteína)/metabolismo
5.
Int J Syst Evol Microbiol ; 70(12): 6067-6078, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33048039

RESUMO

When analysing a large cohort of Staphylococcus haemolyticus, using whole-genome sequencing, five human isolates (four from the skin and one from a blood culture) with aberrant phenotypic and genotypic traits were identified. They were phenotypically similar with yellow colonies, nearly identical 16S rRNA gene sequences and initially speciated as S. haemolyticus based on 16S rRNA gene sequence and MALDI-TOF MS. However, compared to S. haemolyticus, these five strains demonstrate: (i) considerable phylogenetic distance with an average nucleotide identity <95 % and inferred DNA-DNA hybridization <70  %; (ii) a pigmented phenotype; (iii) urease production; and (iv) different fatty acid composition. Based on the phenotypic and genotypic results, we conclude that these strains represent a novel species, for which the name Staphylococcus borealis sp. nov. is proposed. The novel species belong to the genus Staphylococcus and is coagulase- and oxidase-negative and catalase-positive. The type strain, 51-48T, is deposited in the Culture Collection University of Gothenburg (CCUG 73747T) and in the Spanish Type Culture Collection (CECT 30011T).


Assuntos
Sangue/microbiologia , Filogenia , Pele/microbiologia , Staphylococcus/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Humanos , Noruega , Hibridização de Ácido Nucleico , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Staphylococcus/isolamento & purificação
6.
Int J Syst Evol Microbiol ; 70(8): 4544-4554, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32618559

RESUMO

The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Portugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acinetobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA-DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T).


Assuntos
Acinetobacter/classificação , Microbiologia de Alimentos , Carne/microbiologia , Filogenia , Acinetobacter/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Tipagem de Sequências Multilocus , Hibridização de Ácido Nucleico , Portugal , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
7.
Sci Rep ; 10(1): 11656, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669560

RESUMO

We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198T and CCUG 4207T, the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198T and CCUG 4207T are derived from deposit of the same strain at two different culture collections. NCTC 8198T was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207T was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.


Assuntos
Mapeamento Cromossômico , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Streptococcus pyogenes/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodos , Técnicas de Tipagem Bacteriana , Sequência de Bases , Sistemas CRISPR-Cas , DNA Bacteriano/metabolismo , Genoma Bacteriano , Nanoporos , Prófagos/genética , Análise de Sequência de DNA , Streptococcus pyogenes/classificação , Streptococcus pyogenes/virologia , Fatores de Virulência/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-32509595

RESUMO

Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.


Assuntos
Genômica , Streptococcus pneumoniae , Streptococcus , Genótipo , Streptococcus/genética , Streptococcus pneumoniae/genética
9.
Microorganisms ; 8(6)2020 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-32545759

RESUMO

Escherichia coli strain CCUG 78773 is a virulent extended-spectrum ß-lactamase (ESBL)-producing ST131-O25b type strain isolated during an outbreak at a regional university hospital. The complete and closed genome sequence, comprising one chromosome (5,076,638 bp) and six plasmids (1718-161,372 bp), is presented. Characterization of the genomic features detected the presence of 59 potential antibiotic resistance factors, including three prevalent ß-lactamases. Several virulence associated elements were determined, mainly related with adherence, invasion, biofilm formation and antiphagocytosis. Twenty-eight putative type II toxin-antitoxin systems were found. The plasmids were characterized, through in silico analyses, confirming the two ß-lactamase-encoding plasmids to be conjugative, while the remaining plasmids were mobilizable. BLAST analysis of the plasmid sequences showed high similarity with plasmids in E. coli from around the world. Expression of many of the described virulence and AMR factors was confirmed by proteomic analyses, using bottom-up, liquid chromatography-tandem mass spectrometry (LC-MS/MS). The detailed characterization of E. coli strain CCUG 78773 provides a reference for the relevance of genetic elements, as well as the characterization of antibiotic resistance and the spread of bacteria harboring ESBL genes in the hospital environment.

10.
BMC Microbiol ; 20(1): 80, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32264835

RESUMO

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.


Assuntos
Técnicas Bacteriológicas/métodos , Proteínas de Membrana/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus haemolyticus/classificação , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Colágeno/metabolismo , Fibronectinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Espectrometria de Massas , Plásticos/química , Staphylococcus haemolyticus/crescimento & desenvolvimento , Staphylococcus haemolyticus/isolamento & purificação , Staphylococcus haemolyticus/patogenicidade , Simbiose
11.
Mol Cell Proteomics ; 19(3): 518-528, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31941798

RESUMO

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.


Assuntos
Proteínas de Bactérias/metabolismo , Haemophilus influenzae/metabolismo , Moraxella catarrhalis/metabolismo , Peptídeos/metabolismo , Staphylococcus aureus/metabolismo , Streptococcus pneumoniae/metabolismo , Biomarcadores/metabolismo , Haemophilus influenzae/isolamento & purificação , Humanos , Moraxella catarrhalis/isolamento & purificação , Sistema Respiratório/microbiologia , Infecções Respiratórias/microbiologia , Especificidade da Espécie , Staphylococcus aureus/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Espectrometria de Massas em Tandem
12.
Sex Transm Infect ; 96(3): 160-165, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31932359

RESUMO

OBJECTIVE: Internet-based testing for Chlamydia trachomatis (CT) with self-sampling at home has gradually been implemented in Sweden since 2006 as a free-of-charge service within the public healthcare system. This study evaluated the national diagnostic outcome of this service. METHODS: Requests for data on both self-sampling at home and clinic-based sampling for CT testing were sent to the laboratories in 18 of 21 counties. Four laboratories were also asked to provide data on testing patterns at the individual level for the years 2013-2017. RESULTS: The proportion of self-sampling increased gradually from 2013, comprising 22.0% of all CT tests in Sweden in 2017. In an analysis of 14 counties (representing 83% of the population), self-sampling increased by 115% between 2013 and 2017 for women, compared with 71% for men, while test volumes for clinic-based sampling were fairly constant for both sexes (1.8% increase for women, 15% increase for men). In 2017 self-sampling accounted for 20.3% of all detected CT cases, and the detection rate was higher than, but similar to, clinic-based testing (5.5% vs 5.1%). The proportion of self-sampling men was also higher, but similar (33.7% vs 30.8%). Analysis of individual testing patterns in four counties over 5 years showed a higher proportion of men using self-sampling only (67%, n=10 533) compared with women (40%, n=8885). CONCLUSIONS: Self-sampling has increased substantially in recent years, especially among women. This service is at least as beneficial as clinic-based screening for detection of CT, and self-sampling reaches men more than clinic-based testing.


Assuntos
Infecções por Chlamydia/diagnóstico , Testes Diagnósticos de Rotina/métodos , Internet , Autoexame/métodos , Autoexame/estatística & dados numéricos , Manejo de Espécimes/métodos , Manejo de Espécimes/estatística & dados numéricos , Adolescente , Adulto , Chlamydia trachomatis/isolamento & purificação , Feminino , Pesquisa sobre Serviços de Saúde , Humanos , Masculino , Pessoa de Meia-Idade , Suécia , Adulto Jovem
13.
Syst Appl Microbiol ; 43(1): 126039, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776051

RESUMO

Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).


Assuntos
Infecções por Corynebacterium/microbiologia , Corynebacterium/classificação , Microbiologia Ambiental , Proteínas de Bactérias/genética , Composição de Bases , Corynebacterium/química , Corynebacterium/citologia , Corynebacterium/fisiologia , DNA Bacteriano/genética , Ácidos Graxos/química , Tamanho do Genoma , Genoma Bacteriano/genética , Glicolipídeos/química , Humanos , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Especificidade da Espécie , Vitamina K 2/química
14.
Front Microbiol ; 10: 2511, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781055

RESUMO

The family Enterobacteriaceae is a taxonomically diverse and widely distributed family containing many human commensal and pathogenic species that are known to carry transferable antibiotic resistance determinants. Characterization of novel taxa within this family is of great importance in order to understand the associated health risk and provide better treatment options. The aim of the present study was to characterize a Gram-negative bacterial strain (CCUG 66741) belonging to the family Enterobacteriaceae, isolated from a wound infection of an adult patient, in Sweden. Initial phenotypic and genotypic analyses identified the strain as a member of the family Enterobacteriaceae but could not assign it to any previously described species. The complete 16S rRNA gene sequence showed highest similarity (98.8%) to four species. Whole genome sequencing followed by in silico DNA-DNA similarity analysis and average nucleotide identity (ANI) analysis confirmed that strain CCUG 66741 represents a novel taxon. Sequence comparisons of six house-keeping genes (16S rRNA, atpD, dnaJ, gyrB, infB, rpoB) with those of the type strains of the type species of related genera within the family Enterobacteriaceae indicated that the strain embodies a novel species within the family. Phylogenomic analyses (ANI-based and core genome-based phylogeny) showed that strain CCUG 66741 forms a distinct clade, representing a novel species of a distinct, new genus within the family Enterobacteriaceae, for which the name Scandinavium goeteborgense gen. nov., sp. nov. is proposed, with CCUG 66741T as the type strain (= CECT 9823T = NCTC 14286T). S. goeteborgense CCUG 66741T carries a novel variant of a chromosomally-encoded quinolone resistance gene (proposed qnrB96). When expressed in Escherichia coli, the qnrB96 gene conferred five-fold increase in minimum inhibitory concentration against ciprofloxacin. This study highlights the importance and the utility of whole genome sequencing for pathogen identification in clinical settings.

16.
Int J Syst Evol Microbiol ; 69(8): 2268-2276, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31125302

RESUMO

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.


Assuntos
Enterococcaceae/classificação , Pé/microbiologia , Filogenia , Carne Vermelha/microbiologia , Ferimentos e Lesões/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Enterococcaceae/isolamento & purificação , Ácidos Graxos/química , Genes Bacterianos , Humanos , Masculino , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
17.
JCI Insight ; 52019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30920392

RESUMO

Skeletal muscle weakness in patients suffering from rheumatoid arthritis (RA) adds to their impaired working abilities and reduced quality of life. However, little molecular insight is available on muscle weakness associated with RA. Oxidative stress has been implicated in the disease pathogenesis of RA. Here we show that oxidative post-translational modifications of the contractile machinery targeted to actin result in impaired actin polymerization and reduced force production. Using mass spectrometry, we identified the actin residues targeted by oxidative 3-nitrotyrosine (3-NT) or malondialdehyde adduct (MDA) modifications in weakened skeletal muscle from mice with arthritis and patients afflicted by RA. The residues were primarily located to three distinct regions positioned at matching surface areas of the skeletal muscle actin molecule from arthritis mice and RA patients. Moreover, molecular dynamic simulations revealed that these areas, here coined "hotspots", are important for the stability of the actin molecule and its capacity to generate filaments and interact with myosin. Together, these data demonstrate how oxidative modifications on actin promote muscle weakness in RA patients and provide novel leads for targeted therapeutic treatment to improve muscle function.


Assuntos
Actinas/metabolismo , Artrite Reumatoide/metabolismo , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Actinas/química , Animais , Artrite Reumatoide/complicações , Modelos Animais de Doenças , Feminino , Humanos , Malondialdeído , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Dinâmica Molecular , Contração Muscular/fisiologia , Debilidade Muscular/etiologia , Debilidade Muscular/fisiopatologia , Músculo Esquelético/fisiopatologia , Miosinas/química , Miosinas/metabolismo , Polimerização , Processamento de Proteína Pós-Traducional , Tirosina/análogos & derivados
18.
Eur J Clin Microbiol Infect Dis ; 38(3): 505-514, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30707378

RESUMO

Respiratory tract infections (RTI) are more commonly caused by viral pathogens in children than in adults. Surprisingly, little is known about antibiotic use in children as compared to adults with RTI. This prospective study aimed to determine antibiotic misuse in children and adults with RTI, using an expert panel reference standard, in order to prioritise the target age population for antibiotic stewardship interventions. We recruited children and adults who presented at the emergency department or were hospitalised with clinical presentation of RTI in The Netherlands and Israel. A panel of three experienced physicians adjudicated a reference standard diagnosis (i.e. bacterial or viral infection) for all the patients using all available clinical and laboratory information, including a 28-day follow-up assessment. The cohort included 284 children and 232 adults with RTI (median age, 1.3 years and 64.5 years, respectively). The proportion of viral infections was larger in children than in adults (209(74%) versus 89(38%), p < 0.001). In case of viral RTI, antibiotics were prescribed (i.e. overuse) less frequently in children than in adults (77/209 (37%) versus 74/89 (83%), p < 0.001). One (1%) child and three (2%) adults with bacterial infection were not treated with antibiotics (i.e. underuse); all were mild cases. This international, prospective study confirms major antibiotic overuse in patients with RTI. Viral infection is more common in children, but antibiotic overuse is more frequent in adults with viral RTI. Together, these findings support the need for effective interventions to decrease antibiotic overuse in RTI patients of all ages.


Assuntos
Antibacterianos/uso terapêutico , Gestão de Antimicrobianos/normas , Prescrição Inadequada/estatística & dados numéricos , Infecções Respiratórias/tratamento farmacológico , Idoso , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/epidemiologia , Pré-Escolar , Feminino , Humanos , Lactente , Israel/epidemiologia , Masculino , Pessoa de Meia-Idade , Países Baixos/epidemiologia , Estudos Prospectivos , Padrões de Referência , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Viroses/diagnóstico , Viroses/tratamento farmacológico , Viroses/epidemiologia
19.
PLoS One ; 13(12): e0208804, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532202

RESUMO

A range of methodologies may be used for analyzing bacteria, depending on the purpose and the level of resolution needed. The capability for recognition of species distinctions within the complex spectrum of bacterial diversity is necessary for progress in microbiological research. In clinical settings, accurate, rapid and cost-effective methods are essential for early and efficient treatment of infections. Characterization and identification of microorganisms, using, bottom-up proteomics, or "proteotyping", relies on recognition of species-unique or associated peptides, by tandem mass spectrometry analyses, dependent upon an accurate and comprehensive foundation of genome sequence data, allowing for differentiation of species, at amino acid-level resolution. In this study, the high resolution and accuracy of MS/MS-based proteotyping was demonstrated, through analyses of the three phylogenetically and taxonomically most closely-related species of the Mitis Group of the genus Streptococcus: i.e., the pathogenic species, Streptococcus pneumoniae (pneumococcus), and the commensal species, Streptococcus pseudopneumoniae and Streptococcus mitis. To achieve high accuracy, a genome sequence database used for matching peptides was created and carefully curated. Here, MS-based, bottom-up proteotyping was observed and confirmed to attain the level of resolution necessary for differentiating and identifying the most-closely related bacterial species, as demonstrated by analyses of species of the Streptococcus Mitis Group, even when S. pneumoniae were mixed with S. pseudopneumoniae and S. mitis, by matching and identifying more than 200 unique peptides for each species.


Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Proteoma/metabolismo , Proteômica , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/metabolismo , Espectrometria de Massas em Tandem
20.
Eur J Clin Microbiol Infect Dis ; 37(1): 57-68, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28924947

RESUMO

In this study we present a method using whole cell MALDI-TOF MS and VITEK MS RUO/SARAMIS as a rapid epidemiological screening tool. MRSA was used as a model organism for setting up the screening strategy. A collection of well-characterised MRSA strains representing the 19 most common Pulsed-Field Gel Electrophoresis (PFGE)-types in the region of South-West Sweden for the past 20 years was analysed with MALDI-TOF MS. A total of 111 MRSA strains were used for creating 19 PFGE-specific Superspectra using VITEK MS RUO/SARAMIS. Prior to performing the final analysis, the 19 Superspectra were combined into ten groups displaying similar peak patterns, hereafter named "MALDI-types". Two-hundred fifty-five MRSA strains were analysed to test the constructed Superspectra/MALDI-type database. Matches to the Superspectra above a threshold of 65% (corresponding to the number of matched peaks in the Superspectrum) were considered as positive assignment of a strain to a MALDI-type. The median peak matching value for correct assignment of a strain to a MALDI-type was 78% (range 65.3-100%). In total, 172 strains (67.4%) were assigned to the correct MALDI-type and only 5.5% of the strains were incorrectly assigned to another MALDI-type than the expected based on the PFGE-type of the strain. We envision this methodology as a cost-efficient step to be used as a first screening strategy in the typing scheme of MRSA isolates, to exclude epidemiological relatedness of isolates or to identify the need for further typing.


Assuntos
Técnicas de Tipagem Bacteriana/métodos , Monitoramento Epidemiológico , Staphylococcus aureus Resistente à Meticilina/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/epidemiologia , Bases de Dados Factuais , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia
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