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1.
Bratisl Lek Listy ; 121(3): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32115974

RESUMO

BACKGROUND: Clostridium (Clostridioides) difficile is the most common pathogen of nosocomial and antibiotic-related diarrhea in health-care facilities. The aim of the analysis was to show the testing algorithm and to identify hypervirulent strains (suspected RT 027). METHODS: The retrospective analysis of patient samples suspected on CDI was carried out by a two-step algorithm. Biological specimens were analysed by GDH or culture, immunoenzymatic assay on toxins A/B and selected samples also by a real-time PCR. RESULTS: In 1006 specimen suspected on CDI, 202 specimens were evaluated as positive in the two-step algorithm. Conflicting results (64 C. difficile isolates) were tested in a three-step algorithm by a real-time PCR and revealed 59 toxigenic and non RT 027 ribotypes. Statistically significant dependence among the independent variables, such as: diagnostic parameters and length of hospitalization (p = 0.175) and C. difficile (suspected RT027) ribotypes was not found. CONCLUSION: The results of PCR ribotyping showed a high prevalence of hypervirulent and toxigenic ribotypes in the studied sample. A resistance to vancomycin was found in one isolate. The PCR method contributed to the rapid laboratory diagnosis and thus treatment of high risk patients or was used as a third step in in the case of unclear results of standard diagnostic methods(Tab. 1, Fig. 4, Ref. 18). Text in PDF www.elis.sk.


Assuntos
Infecções por Clostridium , Clostridium difficile , Ribotipagem , Algoritmos , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Infecções por Clostridium/diagnóstico , Clostridium difficile/genética , Humanos , Estudos Retrospectivos
2.
Microbiol Res ; 236: 126444, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32169751

RESUMO

Little is known about fluorescent Pseudomonas and investigations are needed to help us better understand how their species work. The aim was here to mimic what naturally occurs in environmental water containing strains isolated from mid-mountain water samples and identified as Pseudomonas fluorescens by conventional biochemical techniques. Three strains were cultured before being directly inoculated into distilled water. Surprisingly, the three cell-less extracts obtained after spinning the bacterial suspensions showed strong in vitro anti-oxidative effects against superoxide anion and hydroxyl radical but with discrepancies. The extracts obtained were found to contain antioxidant proteins among other stress proteins that were released by viable bacteria. They were identified using tandem/mass spectrometry and showed different profiles in sodium-dodecyl sulfate polyacrylamide gel electrophoresis. Bacterial identification was deepened using 16S ribonucleic acid and genome sequencing analyses to explain the differences observed between strains.


Assuntos
Antioxidantes/química , Pseudomonas fluorescens , Antioxidantes/análise , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Biodiversidade , Eletroforese em Gel de Poliacrilamida , Genes Bacterianos , Proteômica , Pseudomonas fluorescens/isolamento & purificação , Pseudomonas fluorescens/metabolismo , RNA Ribossômico 16S , Espectrometria de Massas em Tandem , Água/química
3.
Microbiol Res ; 236: 126468, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32208189

RESUMO

Extracellular proteases from haloarchaea (halolysins) can resist high salt conditions. In this study, the gene encoding a halolysin from Halococcus salifodinae was identified. The hlyA gene encoded an active halolysin with the classical Asp-His-Ser catalytic triad of serine proteases. Site-directed mutagenesis showed that the three cysteine residues in the catalytic domain were important for the extracellular proteolytic activity and displayed an additive effect on the activity. Truncation mutants of the C-terminal extension (CTE) domain displayed very low or almost no extracellular protease activity towards milk and small peptide substrates, indicating its importance for the function of HlyA. CTE can be functionally interchangeable among halolysins. Additionally, the HlyA expressing strain as a starter culture for fish sauce fermentation significantly increased the peptide release and total free amino acid content in fish sauce. This study enriches our knowledge of the key amino acid residues and domains of halolysins, and provides an opportunity for applications of halolysins in fish sauce fermentation.


Assuntos
Halococcus/genética , Serina Endopeptidases , Serina Proteases/biossíntese , Sequência de Aminoácidos/genética , Proteínas de Bactérias/análise , Biotecnologia , Domínio Catalítico/genética , Fermentação , Produtos Pesqueiros , Serina Endopeptidases/biossíntese , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Proteases/química , Serina Proteases/genética
4.
Ann Lab Med ; 40(3): 259-263, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31858767

RESUMO

There is an urgent need for accurate and rapid diagnostic assays capable of identifying carbapenemase-producing Enterobacteriaceae (CPE). We assessed the performance of the RESIST-4 O.K.N.V. (OKNV) assay (Coris BioConcept, Gembloux, Belgium) for the identification of oxacillinase (OXA)-48-like-, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-ß-lactamase (NDM)-, and Verona integron-encoded metallo-ß-lactamase (VIM)-producing Enterobacteriaceae grown on sheep blood agar (SBA) and the CHROMagar KPC medium. Sixty-five carbapenem-resistant Enterobacteriaceae (CRE) isolates with characterized carbapenemase content were used to evaluate the OKNV assay. The assay correctly identified all 30 isolates that produced one of the four targeted carbapenemase families. Additionally, it correctly identified 15 isolates that co-produced KPC and NDM, VIM and NDM or OXA-48-like and NDM, but failed to identify an NDM-1 and OXA-232 co-producing Klebsiella pneumoniae isolate. All 16 non-carbapenemase-producing CRE and four CPE isolates exhibited negative results, and no cross-reaction was observed. Overall, the sensitivity and specificity of the assay were 97.8% and 100%, respectively. The OKNV assay is an accurate and rapid assay for identifying OXA-48-like, KPC, NDM, and VIM carbapenemases produced by Enterobacteriaceae isolates cultured on both SBA and the CHROMagar KPC media in the clinical microbiology laboratory.


Assuntos
Proteínas de Bactérias/análise , Enterobacteriaceae/enzimologia , beta-Lactamases/análise , Técnicas Bacteriológicas/métodos , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Humanos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade
5.
J Fish Dis ; 43(1): 81-89, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31701546

RESUMO

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Doenças dos Peixes/diagnóstico , Carpa Dourada , Infecções por Mycobacterium/veterinária , Mycobacterium/fisiologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Doenças dos Peixes/microbiologia , Mycobacterium/química , Mycobacterium/genética , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/microbiologia , Filogenia , Alinhamento de Sequência
6.
Diagn Microbiol Infect Dis ; 96(1): 114912, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704067

RESUMO

We evaluated the performance of five phenotypic tests [Modified Hodge Test (MHT); combined-disk test (CDT) using phenylboronic acid, EDTA, and cloxacillin; CarbaNP and CarbAcinetoNP; Blue-Carba, Carbapenembac™ and Carbapenembac Metallo™] for carbapenemase detection in Gram-negative bacilli (GNB). A total of 73 carbapenemase producers and 27 non-carbapenemase producers were tested. All GNB were subcultured onto Müeller-Hinton agar (MHA), MacConkey agar (MAC), and sheep blood agar (SBA). High sensitivity (100%) and specificity (100%) was observed for MHA using CarbaNP, Blue-Carba, and Carbapenembac™. The sensitivity and specificity of CarbaNP (98.6%/100%), Blue-Carba (97.1%/91.0%), and Carbapenembac™ (100%/96.5%) were slightly lower for SBA. In contrast, unacceptable sensitivity rates of CarbaNP (71.1%) and Blue-Carba (66.6%), but not Carbapenembac™ (97.3%), were observed for MAC. The colorimetric methods showed high sensitivity and specificity to detect carbapenemase production from isolates grown on MHA or SBA. However, colonies obtained from MAC must not be tested for carbapenemase detection by colorimetric methods.


Assuntos
Ágar/química , Proteínas de Bactérias/análise , Meios de Cultura/química , Enterobacteriaceae/crescimento & desenvolvimento , beta-Lactamases/análise , Antibacterianos/farmacologia , Carbapenêmicos/farmacologia , Colorimetria , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Testes de Sensibilidade Microbiana , Fenótipo , Sensibilidade e Especificidade
7.
Lett Appl Microbiol ; 70(1): 42-47, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31642085

RESUMO

The increasing frequency of class A KPC enzymes, class B metallo-ß-lactamases (MBLs) and class D OXA-48 enzymes in Enterobacteriaceae makes their early identification urgent. A simple commercial MASTDISCS combi Carba plus disc system (MAST-Carba plus) was designed for the identification of MBLs, KPC and OXA-48 carbapenemase genes in Enterobacteriaceae. To validate the MAST-Carba plus, a total of 77 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 84 isolates of noncarbapenemase-producing Enterobacteriaceae (non-CPE) were selected for differentiation of the genes of Enterobacteriaceae by MAST-Carba plus. Meanwhile, the carbapenemase genes such as blaKPC , blaIMP , blaVIM , blaNDM-1 and blaOXA-48 were detected by PCR (polymerase chain reaction). Thus, when considered on the basis of PCR results, the sensitivity of MAST-Carba plus detection of KPC strains is 82·3%, the specificity is 100·0%, the positive predictive value is 100·0% and the negative predictive value is 92·4%. For MBLs strains, the sensitivity is 100·0%, the specificity is 97·1%, the positive predictive value is 84·6% and the negative predictive value is 100·0%. For OXA-48 strains, the sensitivity is 100·0%, the specificity is 99·4%, the positive predictive value is 80·0% and the negative predictive value is 100·0%. Our findings suggest that MAST-Carba plus is a rapid and promising method for identifying the MBLs, KPC and OXA-48 carbapenemase genes in Enterobacteriaceae, which could be exploited in basic microbiology laboratory to prevent the transmission of CPE. SIGNIFICANCE AND IMPACT OF THE STUDY: Not only detection of carbapenemases but also identification of their genes accurately and rapidly in Enterobacteriaceae is still a major challenge for clinical laboratories in order to prevent the transmission of carbapenemase-producing Enterobacteriaceae (CPE). Therefore, this study aimed to evaluate the performance of a new rapid method (MASTDISCS combi Carba plus) for the identification of metallo-ß-lactamases (MBLs), KPC and OXA-48 carbapenemase genes in Enterobacteriaceae clinical isolates.


Assuntos
Proteínas de Bactérias/análise , Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Ensaios Enzimáticos/métodos , beta-Lactamases/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterobacteriaceae/classificação , Enterobacteriaceae/genética , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , beta-Lactamases/genética , beta-Lactamases/metabolismo
8.
PLoS One ; 14(12): e0226576, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31869349

RESUMO

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°Ð¡ behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.


Assuntos
Bacillus subtilis/enzimologia , Desinfecção/métodos , Escherichia coli/enzimologia , Temperatura Alta , Luciferases Bacterianas/química , Redobramento de Proteína , Adenosina Trifosfatases/análise , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/análise , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/análise , Proteínas de Choque Térmico HSP70/análise , Temperatura Alta/uso terapêutico , Cinética , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Viabilidade Microbiana , Chaperonas Moleculares/análise , Organismos Geneticamente Modificados
9.
Anal Bioanal Chem ; 411(29): 7737-7745, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31713014

RESUMO

A newly developed molecularly imprinted photonic polymer (MIPP) film, which was prepared by colloidal crystal templating and surface molecular imprinting, was used for selective capture of S-layer protein (SLP) from a complex Lactobacillus acidophilus sample. The colloidal crystal templates were formed by a dipping process followed by chemical binding of the imprinted template SLP molecules. A sandwich structure consisting of two glass slides was formed after the SLP-silica layer had been covered with a poly(methyl methacrylate) glass slide. After polymerization of the SLP-silica layer with the preprepared polymerization solution, hydrofluoric acid and acetic phosphate buffer solutions removed the silica particles and SLP molecules, respectively. The MIPP film obtained exhibited a three-dimensional, highly ordered and interconnected macroporous structure (pore size greater than 200 nm), which is specifically accessible to SLP molecules. The adsorbed SLP molecules were simply and straightforwardly detected by a fiber-optic spectrometer. The redshift of the Bragg diffraction peak of the MIPP film was linearly related to the number of SLP molecules that had been harvested in the film. The detection limit of the SLP-MMIP-fiber-optic spectrometer method for SLP was 1 ng mL-1. The MIPP sensor was successfully applied to detect SLP molecules in a crudely extracted Lactobacillus acidophilus sample. Our results prove the applicability of the SLP-MIPP film for fast and real-time measurement of SLP. Graphical abstract.


Assuntos
Proteínas de Bactérias/análise , Tecnologia de Fibra Óptica , Glicoproteínas de Membrana/análise , Impressão Molecular , Fótons , Polímeros/química , Análise Espectral/instrumentação , Lactobacillus acidophilus/química , Limite de Detecção
10.
J Med Microbiol ; 68(12): 1723-1731, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31746726

RESUMO

Introduction. Carbapenems are often described as the most effective weapon against infections caused by multidrug-resistant bacteria especially those belonging to the group of non-fermenting bacteria such as Pseudomonas. The main mechanisms leading to resistance are the hyperexpression of certain efflux pumps belonging to the resisto-nodular division and the lower expression of the transmembrane porin OprD, sometimes in combination with excessive production of the intrinsic AmpC. Carbapenemases are assumed to play a secondary role.Aim. The aim of this study was to determine the exact mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from the largest Bulgarian University hospital 'St. George'- Plovdiv.Methodology. A total of 32 clinical isolates collected from different patients' samples resistant to imipenem and/or meropenem were examined via phenotypic and molecular-genetic tests.Results. No metallo-enzyme production was detected. Three isolates were positive for OXA-50-encoding genes in two of them in combination with other oxacillinases or the bla VEB-1 gene. For the first time, OXA-50-producing P. aeruginosa have been reported in Bulgaria. The increased expression or hyperexpression of MexXY-OprM efflux pump was observed as the main mechanism of resistance. In most cases, it was combined with lower expression or lack of OprD with or without MexAB-OprM hyperexpression. No excessive production of AmpC was detected in comparison to the reference ATCC 27853 P. aeruginosa strain.Conclusion. The increased expression or overexpression of MexXY-OprM efflux pumps is the leading cause of carbapenem resistance in our isolates Pseudomonas, detected in 94 % of the bacteria investigated.


Assuntos
Proteínas de Bactérias/análise , Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Porinas/fisiologia , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/análise , beta-Lactamases/fisiologia , Farmacorresistência Bacteriana , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia
11.
Int J Mol Sci ; 20(18)2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31540359

RESUMO

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by 'Candidatus Phytoplasma mali' (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called "Protein in Malus Expressed 2" (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.


Assuntos
Proteínas de Bactérias/genética , Malus/microbiologia , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Tabaco/microbiologia , Sequência de Aminoácidos , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Sobrevivência Celular , Expressão Gênica , Interações Hospedeiro-Patógeno , Malus/citologia , Phytoplasma/química , Phytoplasma/genética , Phytoplasma/patogenicidade , Protoplastos/citologia , Protoplastos/microbiologia , Alinhamento de Sequência , Tabaco/citologia , Fatores de Virulência/análise , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
12.
J Vet Med Sci ; 81(10): 1400-1408, 2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31462609

RESUMO

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.


Assuntos
Proteínas de Bactérias/análise , Mycobacterium bovis/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Tuberculose Bovina/diagnóstico , Animais , Proteínas de Bactérias/isolamento & purificação , Bovinos
13.
Ticks Tick Borne Dis ; 10(6): 101267, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31444126

RESUMO

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results.


Assuntos
Anaplasma phagocytophilum/genética , Anaplasmose/diagnóstico , Búfalos , Doenças das Cabras/diagnóstico , Anaplasmose/microbiologia , Animais , Proteínas de Bactérias/análise , Feminino , Doenças das Cabras/microbiologia , Cabras , Masculino , Tipagem de Sequências Multilocus/veterinária , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Suíça
14.
Comput Biol Chem ; 83: 107110, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31445418

RESUMO

Salmonella, an Enterobacteria is a therapeutically important pathogen for the host. The advancement of genome sequencing of S. enterica serovar Enteritidis have identified a distinct ROD9 pathogenic island, imparting virulence. The occurrence of 17 ROD9 hypothetical proteins, necessitates subsequent bioinformatics approach for structural and functional aspects of protein-protein relations or networks in different pathogenic phenotypes express. A collective analysis using predictive bioinformatics tools that includes NCBI-BLASTp and BLAST2GO annotated the motif patterns and functional significance. The VFDB identified 10 virulence proteins at both genomic and metagenomic level. Phylogenetic analysis revealed a divergent and convergent relationship between 17 ROD9 and 41 SP-1 proteins. Here, combining a comprehensive approach from sequence based, motif recognitions, domain identification, virulence ability to structural modelling provides a precise function to ROD9 proteins biological network, for which no experimental information is available.


Assuntos
Proteínas de Bactérias/análise , Anotação de Sequência Molecular , Salmonella enterica/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bases de Dados de Proteínas , Fenótipo , Salmonella enterica/metabolismo
15.
Rev. esp. quimioter ; 32(4): 370-374, ago. 2019. tab
Artigo em Espanhol | IBECS | ID: ibc-ET5-83

RESUMO

INTRODUCCIÓN: En los últimos años se ha producido un incremento de las infecciones producidas por Staphylococcus aureus resistente a meticilina (SARM). En comparación con las producidas por S. aureus sensible a meticilina (SASM), las infecciones por SARM requieren estancias hospitalarias más prolongadas y presentan mayor mortalidad. La detección rápida de la resistencia a la meticilina por la adquisición del gen mecA que codifica la proteína fijadora de penicilina (PBP2a) es crucial para evitar la diseminación nosocomial e instaurar una correcta terapia antimicrobiana. Nos proponemos evaluar el test inmunocromatográfico rápido para la detección de PBP2a directamente de colonias de S. aureus, PBP2a SA Culture Colony Test(R) (ICPBP2a). MATERIAL Y MÉTODOS: En 107 cepas de S. aureus se estudió la resistencia a meticilina mediante las siguientes pruebas: el sistema automatizado Vitek2(R) (bioMérieux), CHROMagar MRSA II(R) (BD Becton Dickinson ), difusión con disco de cefoxitina, la ICPBP2a (AlereTM) y como método de referencia, la detección molecular del gen mecA. RESULTADOS: La sensibilidad y especificidad para las pruebas de detección fueron para la difusión en agar con disco de cefoxitina 100% y 100% respectivamente, Vitek2(R) 100% y 100%, CHROMagarTM MRSA II 100% y 96%, y la ICPBP2a 98,25% y 100%. CONCLUSIÓN: La inmunocromatografía para la detección de PBP2a es una técnica rápida, fácil y económica. Resulta muy útil para el manejo de brotes hospitalarios


BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen causing both healthcare-associated and community-acquired infection. Rapid and accurate detection of this pathogen is crucial for the use of appropriate antimicrobial therapy and the control of nosocomial spread. METHODS: A total of 107 S. aureus strains were assayed for methicillin resistance: Vitek2(R) (bioMérieux), CHROMagarTM MRSA II (BD Becton Dickinson), disk diffusion in agar for cefoxitin 30 μg and immunochromatography PBP2a SA Culture Colony Test (AlereTM). The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. RESULTS: Sensitivity and specificity were: disk diffusion for cefoxitin 100% and 100% respectively, Vitek2(R) 100 and 100%, CHROMagarTM MRSA II 100 and 96%, and ICPBP2a detection 98,25% and 100%. CONCLUSION: ICPBP2a Culture Colony Test (AlereTM) is fast, efficient and economical technique for detection of penicillin binding protein 2a (PBP2a) from isolates. This assay is a useful tool for the management of hospital outbreaks


Assuntos
Humanos , Técnicas Bacteriológicas/métodos , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Cefoxitina/farmacologia , Meios de Cultura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Imunoensaio/métodos , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/análise , Proteínas de Ligação às Penicilinas/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
16.
Braz J Microbiol ; 50(4): 979-984, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31352634

RESUMO

Control of brucellosis as a worldwide zoonotic disease is based on vaccination of animals and diagnosis of infected cases to be eradicated. Accurate and rapid detection of infected animals is of critical importance for preventing the spread of disease. Current detection of brucellosis is based on whole-cell antigens and investigating serum antibodies against Brucella lipopolysaccharide (LPS). The critical disadvantage is misdiagnosis of vaccinated animals as infected ones and also cross-reactions with other Gram-negative bacteria. Recombinant outer membrane protein 2b (Omp2b) of Brucella abortus was evaluated as a novel serodiagnostic target in comparison to conventional tests which are based on LPS. Recombinant Omp2b (rOmp2b) was expressed in Escherichia coli BL21 and purified by Ni2+-based chromatography. rOmp2b was evaluated in an indirect enzyme-linked immunosorbent assay (ELISA) system for diagnosis of brucellosis, with sera from Brucella-infected mice along with negative sera and sera from mice which were inoculated with other Gram-negative species for assurance of specificity. Thereafter, cattle sera collected from different regions were assessed along with known negative and known positive serum samples. We found that Omp2b can discriminate between Brucella-infected animals and non-infected ones. Results for assessment of two hundred and fifty cattle sera by Omp2b-based indirect ELISA which were compared to Rose Bengal plate agglutination test (RBPT) and serum tube agglutination test (SAT) showed that our proposed procedure has the sensitivity of 88.5%, specificity of 100%, and accuracy of 90.8%. We suggest that recombinant Omp2b could be used as a protein antigen for diagnosis of brucellosis in domestic animals and can be evaluated for detection of human brucellosis.


Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Brucella abortus/isolamento & purificação , Brucelose/veterinária , Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Porinas/análise , Testes Sorológicos/métodos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Brucella abortus/genética , Brucella abortus/imunologia , Brucelose/diagnóstico , Brucelose/microbiologia , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Porinas/genética , Porinas/imunologia
17.
Acta Biochim Pol ; 66(3): 321-327, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31329404

RESUMO

Expression proteomics approaches do not only directly confirm protein coding genes of sequenced genomes but also facilitate resolution of minute qualitative protein differences and improve the quality of genome annotation. Despite development of many tools, use of 2-DE coupled with MS in proteomics is not uncommon. With an accelerated trend of genome sequencing of microorganisms, proteome analysis of animal pathogens with 2-DE has gained more attention in the last decade. Therefore, in this study primarily the protein extraction, sample preparation and loading, IPG strip rehydration, IEF, and SDS-PAGE conditions were improved for high throughput resolution and reproducible 2-DE map of proteins of Mycoplasma bovis HB0801 (M. bovis HB0801- Chinese Strain), a pneumonia pathogen in feedlot cattle, and its attenuated strains. Literally, higher amount of proteins was extracted exploiting the French pressure cell coupled with TCA precipitation when compared to the sonication method. Total protein concentration was determined using a 2D quant Kit. About 330-380 µg TCA treated protein sample, solubilized in calibrated rehydration solution, loaded on 17 cm IPG gel strip (pH 3-10 NL) followed by active rehydration at 50V and isoelectric focusing at final 10 000 Volt (33 uA/gel strip) for 80kVh had revealed well resolved proteins spots on 10% gel stained by CBB R250 (0.15%), representing 83-89% of the total protein coding genes of M. bovis HB0801, estimated by PD Quest (Bio-Rad, USA). Conclusively, this effort attempted to provide more precise 2-DE platform and suitable conditions, after extensive calibration, for future comprehensive proteome and immunoproteome analyses and future research on the elucidation of factors related to pathogenesis of M. bovis in cattle.


Assuntos
Proteínas de Bactérias/análise , Eletroforese em Gel Bidimensional/métodos , Mycoplasma bovis/química , Mycoplasma bovis/isolamento & purificação , Proteoma/análise , Proteômica/métodos , Animais , Sequência de Bases , Bovinos , Eletroforese em Gel de Poliacrilamida/métodos , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/métodos , Espectrometria de Massas , Infecções por Mycoplasma/microbiologia , Pneumonia/microbiologia
18.
Ticks Tick Borne Dis ; 10(6): 101260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31327747

RESUMO

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.


Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/epidemiologia , Camelus , Ixodidae/microbiologia , Infestações por Carrapato/veterinária , Anaplasma/genética , Anaplasmose/microbiologia , Animais , Proteínas de Bactérias/análise , Sequência de Bases , Chaperonina 60/análise , DNA Bacteriano/análise , Feminino , Ixodidae/fisiologia , Masculino , Filogenia , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Infestações por Carrapato/epidemiologia , Tunísia/epidemiologia
19.
Rev Esp Quimioter ; 32(4): 370-374, 2019 Aug.
Artigo em Espanhol | MEDLINE | ID: mdl-31293115

RESUMO

OBJECTIVE: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen causing both healthcare-associated and community-acquired infection. Rapid and accurate detection of this pathogen is crucial for the use of appropriate antimicrobial therapy and the control of nosocomial spread. METHODS: A total of 107 S. aureus strains were assayed for methicillin resistance: Vitek2® (bioMérieux), CHROMagarTM MRSA II (BD Becton Dickinson), disk diffusion in agar for cefoxitin 30 µg and immunochromatography PBP2a SA Culture Colony Test (AlereTM). The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. RESULTS: Sensitivity and specificity were: disk diffusion for cefoxitin 100% and 100% respectively, Vitek2® 100 and 100%, CHROMagarTM MRSA II 100 and 96%, and ICPBP2a detection 98,25% and 100%. CONCLUSIONS: ICPBP2a Culture Colony Test (AlereTM) is fast, efficient and economical technique for detection of penicillin binding protein 2a (PBP2a) from isolates. This assay is a useful tool for the management of hospital outbreaks.


Assuntos
Técnicas Bacteriológicas/métodos , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/microbiologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Antibacterianos/farmacologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Cefoxitina/farmacologia , Meios de Cultura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Humanos , Imunoensaio/métodos , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/análise , Proteínas de Ligação às Penicilinas/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
20.
J Vet Diagn Invest ; 31(5): 747-751, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31272304

RESUMO

We report herein Rhodococcus equi infection in an 11-y-old, male llama with a history of diarrhea and endoparasitism. Postmortem examination revealed granulomatous and ulcerative enteritis, pyogranulomatous mesenteric lymphadenitis, fibrinosuppurative peritonitis, and granulomatous hepatitis. Intralesional macrophages were laden with gram-positive cocci. Bacteriology identified R. equi, and cultures tested positive for R. equi choE and vapA genes by PCR. This case expands the reported spectrum of lesions associated with R. equi infections in llamas from pyogranulomatous bronchopneumonia and peripheral lymphadenitis to pyogranulomatous mesenteric lymphadenitis and enteritis. We also link a R. equi that is carrying the virulent-associated protein gene VapA to clinical disease in New World camelids.


Assuntos
Infecções por Actinomycetales/veterinária , Camelídeos Americanos , Enterite/veterinária , Linfadenite Mesentérica/veterinária , Rhodococcus equi/isolamento & purificação , Infecções por Actinomycetales/microbiologia , Animais , Animais Domésticos , Proteínas de Bactérias/análise , Enterite/microbiologia , Masculino , Linfadenite Mesentérica/microbiologia , Oregon , Rhodococcus equi/genética , Rhodococcus equi/patogenicidade , Fatores de Virulência
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