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1.
Ann Agric Environ Med ; 26(3): 392-395, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31559791

RESUMO

Existing research for using the protective antigen (PA) of Bacillus anthracis as a vaccine component shows that protection against anthrax may be obtained using fragments of this protein. The aim of the research is to check whether the selected protein fragment of the protective antigen (domain 4) encoded by an appropriate nucleotide sequence of gene pag of B. anthracis, was expressed in the bacterial system of E. coli. In order to examine the selected sequence of the pag gene, a PCR reaction and a highly effective TOPO cloning strategy were used, followed by purification of the recombinant proteins and their detection by a western-blot method. In the planning of the PA4 antigen expression a higher level of effectiveness in production of small protein - domain 4 - was anticipated. As a result, the 139 amino acids protein fragment of B. anthracis PA (domain 4) was isolated. The research may have found the basis for in vivo research aimed at finding potential anthrax vaccine components.


Assuntos
Vacinas contra Antraz/imunologia , Antraz/microbiologia , Antígenos de Bactérias/imunologia , Bacillus anthracis/imunologia , Toxinas Bacterianas/imunologia , Animais , Antraz/imunologia , Antraz/prevenção & controle , Vacinas contra Antraz/administração & dosagem , Vacinas contra Antraz/genética , Vacinas contra Antraz/isolamento & purificação , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/química , Bacillus anthracis/genética , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Domínios Proteicos
2.
N Z Vet J ; 67(6): 329-332, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31378159

RESUMO

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.


Assuntos
Adenocarcinoma/veterinária , Toxinas Bacterianas/genética , Neoplasias Intestinais/veterinária , Metaloendopeptidases/genética , Doenças dos Ovinos/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/microbiologia , Animais , Toxinas Bacterianas/isolamento & purificação , DNA Bacteriano/análise , Genes Bacterianos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/microbiologia , Metaloendopeptidases/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologia
3.
Microb Pathog ; 134: 103600, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31202906

RESUMO

INTRODUCTION: Severe intestinal infections caused by V. cholerae, ETEC and EHEC have contributed to the mortality rate in developing countries. Vibrio Cholera, ETEC and EHEC bacterium with the production of CT, LT and Stx2 toxins respectively lead to severe watery and bloody diarrhea. This study aimed to investigate a trimeric vaccine candidate containing recombinant chimeric protein, encapsulate the protein in chitosan nanoparticles and assess its immunogenicity. METHODS: The LSC recombinant gene was used. It is composed of LTB (L), STXB (S) and CTXB (C) subunits respectively. The LSC recombinant protein was expressed and purified and confirmed by western blotting. The purified protein was encapsulated in chitosan nanoparticles, and its size was measured. BalB/c mice were immunized in four groups through oral and injection methods by LSC protein. The antibody titer was then evaluated by ELISA, and finally, the challenge test of the toxins from all three bacteria was done on the immunized mouse. RESULTS: After expression and purification LSC protein size of nanoparticles containing protein was measured at 104.6 nm. Nanoparticles were able to induce systemic and mucosal immune responses by generating a useful titer of IgG and IgA. The challenge results with LT, CT and Stx toxins showed that the LSC protein might partially neutralize the effect of toxins. CONCLUSION: LSC chimeric protein with the simultaneous three essential antigens have a protective effect against the toxins produced by ETEC, EHEC and Vibrio cholera bacteria and it can be used in vaccines to prevent Diarrhea caused by these three bacteria.


Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Quitosana/farmacologia , Imunização , Nanopartículas/química , Proteínas Recombinantes de Fusão/imunologia , Vacinação , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Toxina da Cólera/genética , Toxina da Cólera/imunologia , Diarreia/microbiologia , Diarreia/prevenção & controle , Modelos Animais de Doenças , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/imunologia , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/imunologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Regulação Bacteriana da Expressão Gênica , Imunidade nas Mucosas , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Proteínas Recombinantes de Fusão/genética , Toxinas Shiga/genética , Toxinas Shiga/imunologia , Análise de Sobrevida , Vibrio cholerae/genética , Vibrio cholerae/imunologia
4.
Microb Pathog ; 134: 103593, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31195111

RESUMO

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to ß-lactam and non-ß-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.


Assuntos
Toxinas Bacterianas/genética , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/genética , Escherichia coli Uropatogênica/genética , Fatores de Virulência/genética , Adesinas de Escherichia coli/genética , Animais , Toxinas Bacterianas/classificação , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/isolamento & purificação , Linhagem Celular , Clonagem Molecular , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/imunologia , Proteínas de Escherichia coli/isolamento & purificação , Feminino , Regulação Bacteriana da Expressão Gênica , Aptidão Genética , Variação Genética , Humanos , Testes de Sensibilidade Microbiana , Fenótipo , Filogenia , Coelhos , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Virulência/genética
5.
Nat Commun ; 10(1): 2641, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201325

RESUMO

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.


Assuntos
Toxinas Bacterianas/química , Clostridium perfringens/ultraestrutura , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Biotecnologia/métodos , Linhagem Celular , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controle , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Clostridium perfringens/patogenicidade , Microscopia Crioeletrônica , Cães , Enterotoxemia/microbiologia , Enterotoxemia/prevenção & controle , Modelos Moleculares , Mutagênese Sítio-Dirigida , Nanotecnologia/métodos , Conformação Proteica em Folha beta/genética , Multimerização Proteica/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 194-202, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31059926

RESUMO

A rapid and sensitive liquid chromatography-mass spectrometry assay was developed and used to quantify emetic cereulide peptide exotoxin, which can be related to possible Bacillus cereus contamination in monoclonal antibody (mAb) bioprocess feeds. The assay limit of detection was 0.05 ng/mL (~1 fmol injected) and limit of quantification 0.16 ng/mL (~3 fmol injected) over a standard curve with >3 orders of magnitude linear dynamic range. The assay allowed quantification of toxin removal in an established two-step mAb purification process consisting of Protein A affinity chromatography followed by multi-modal anion exchange chromatography. Toxin content was ascertained in process stream sample fractions as well as on the Protein A affinity column. An optimized analytical method allowed separation of cereulide toxin from other mAb cell culture components within 6 min. Spiking experiments showed that samples should be collected in high (80% v/v) content acetonitrile to reduce nonspecific losses of the cereulide. The majority of mAb purification process-associated cereulide was detected in the Protein A flow through fraction, whereas only residual amounts were found in wash, strip, and elution fractions. Column cleaning-in-place (CIP) procedures were evaluated to prevent carryover between affinity capture cycles. No carryover was detected between cycles, however trace amounts of cereulide were extracted from the Protein A resin. Increasing the CIP NaOH concentration from 0.1 M to 0.5 M, and contact time from 15 min to 1 h, improved removal of residual cereulide from the resin. Applicability of CIP clearance of cereulide during Protein A chromatography was confirmed with three different mAb feeds. Post Protein A polishing, via target flow through on a multi-modal anion exchange chromatography column, resulted in a product pool with no detectable cereulide. Approximately 5 logs of reduction in cereulide concentration was obtained over the two-step chromatography process. Cereulide contamination is well known and of concern in food processing, however this research may be the first LC-MS quantification of cereulide contamination, and its clearance, in biopharmaceutical mAb processing. The analytical method may also be used to rapidly screen for cereulide contamination in upstream cell culture process streams, prior to downstream product purification. This will allow appropriate measures to be taken to reduce toxin exposure to downstream bioprocess raw materials, consumables and equipment.


Assuntos
Anticorpos Monoclonais/química , Toxinas Bacterianas/isolamento & purificação , Cromatografia de Afinidade/métodos , Depsipeptídeos/isolamento & purificação , Proteína Estafilocócica A/metabolismo , Animais , Bacillus cereus , Células CHO , Cricetinae , Cricetulus , Depsipeptídeos/metabolismo , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes
7.
Int J Biol Macromol ; 134: 1120-1131, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31129209

RESUMO

Immunotoxins are a class of recombinant proteins which consist of an antibody and a part of a bacterial or herbal toxin. Immunotoxins containing Pseudomonas aeruginosa exotoxin A (PEA) have been found to be very applicable in clinical trials. Many obstacles such as solubility and absorbency reduce their usability in solid tumors. The current study aims to overcome the mentioned barriers by addition and removal of functional and non-functional domains with a structural approach. In the experimental section, we took advantage of molecular dynamics simulations to predict the functionality of candidate immunotoxins which target human HER2 receptors and confirmed our findings with in vitro experiments. We found out when no changes were made to domain II of PEA, addition of solubilizing domains to immunotoxins would not reduce their targeting and anti-tumor activity, while increasing the yield of expression and stability. On the other side, when we replaced domain II with eleven amino acids of furin cleavage site (FCS), the activity of the immunotoxin was mainly affected by the FCS neighboring domains and linkers. A combination of seven beneficial point mutations in domain III was also assessed and reconfirmed that the toxicity of the immunotoxin would be reduced dramatically. The obtained results indicate that the addition or removal of domains cannot depict the activity of immunotoxins and the matter should be assessed structurally in advance.


Assuntos
ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Imunotoxinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Fatores de Virulência/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/isolamento & purificação , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Ciclo Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Exotoxinas/química , Exotoxinas/isolamento & purificação , Humanos , Imunotoxinas/química , Imunotoxinas/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão , Solubilidade , Relação Estrutura-Atividade , Sumoilação , Fatores de Virulência/química , Fatores de Virulência/isolamento & purificação
8.
J Mol Model ; 25(5): 136, 2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31028552

RESUMO

Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/ß architecture, in which a single ß sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25 kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.


Assuntos
Bacillus thuringiensis/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Expressão Gênica , Proteínas Recombinantes , Sequência de Aminoácidos , Toxinas Bacterianas/isolamento & purificação , Clonagem Molecular , Amplificação de Genes , Simulação de Dinâmica Molecular , Plasmídeos/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade
9.
J Microbiol ; 57(6): 450-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31012060

RESUMO

Next-generation DNA sequencing technology was applied to generate molecular data from semiarid reservoirs during well-defined seasons. Target sequences of 16S-23S rRNA ITS and cpcBA-IGS were used to reveal the taxonomic groups of cyanobacteria present in the samples, and genes coding for cyanotoxins such as microcystins (mcyE), saxitoxins (sxtA), and cylindrospermopsins (cyrJ) were investigated. The presence of saxitoxins in the environmental samples was evaluated using ELISA kit. Taxonomic analyses of high-throughput DNA sequencing data showed the dominance of the genus Microcystis in Mundaú reservoir. Furthermore, it was the most abundant genus in the dry season in Ingazeira reservoir. In the rainy season, 16S-23S rRNA ITS analysis revealed that Cylindrospermopsis raciborskii comprised 46.8% of the cyanobacterial community in Ingazeira reservoir, while the cpcBAIGS region revealed that C. raciborskii (31.8%) was the most abundant taxon followed by Sphaerospermopsis aphanizomenoides (17.3%) and Planktothrix zahidii (16.6%). Despite the presence of other potential toxin-producing genera, the detected sxtA gene belonged to C. raciborskii, while the mcyE gene belonged to Microcystis in both reservoirs. The detected mcyE gene had good correlation with MC content, while the amplification of the sxtA gene was related to the presence of STX. The cyrJ gene was not detected in these samples. Using DNA analyses, our results showed that the cyanobacterial composition of Mundaú reservoir was similar in successive dry seasons, and it varied between seasons in Ingazeira reservoir. In addition, our data suggest that some biases of analysis influenced the cyanobacterial communities seen in the NGS output of Ingazeira reservoir.


Assuntos
Biodiversidade , Cianobactérias/classificação , Cianobactérias/isolamento & purificação , Água Potável/microbiologia , Análise de Sequência de DNA/métodos , Microbiologia da Água , Abastecimento de Água , Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Brasil , Cianobactérias/genética , DNA Bacteriano/análise , Monitoramento Ambiental/métodos , Genes Bacterianos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microcystis/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Saxitoxina/genética , Estações do Ano , Uracila/análogos & derivados
10.
Toxins (Basel) ; 11(4)2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987300

RESUMO

Mycolactone, the amphiphilic macrolide toxin secreted by Mycobacterium ulcerans, plays a significant role in the pathology and manifestations of Buruli ulcer (BU). Consequently, it follows that the toxin is a suitable target for the development of diagnostics and therapeutics for this disease. Yet, several challenges have deterred such development. For one, the lipophilic nature of the toxin makes it difficult to handle and store and contributes to variability associated with laboratory experimentation and purification yields. In this manuscript, we have attempted to incorporate our understanding of the lipophilicity of mycolactone in order to define the optimal methods for the storage, handling, and purification of this toxin. We present a systematic correlation of variability associated with measurement techniques (thin-layer chromatography (TLC), mass spectrometry (MS), and UV-Vis spectrometry), storage conditions, choice of solvents, as well as the impact of each of these on toxin function as assessed by cellular cytotoxicity. We also compared natural mycolactone extracted from bacterial culture with synthesized toxins in laboratory (solvents, buffers) and physiologically relevant (serum) matrices. Our results point to the greater stability of mycolactone in organic, as well as detergent-containing, solvents, regardless of the container material (plastic, glass, or silanized tubes). They also highlight the presence of toxin in samples that may be undetectable by any one technique, suggesting that each detection approach captures different configurations of the molecule with varying specificity and sensitivity. Most importantly, our results demonstrate for the very first time that amphiphilic mycolactone associates with host lipoproteins in serum, and that this association will likely impact our ability to study, diagnose, and treat Buruli ulcers in patients.


Assuntos
Toxinas Bacterianas , Macrolídeos , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Camada Delgada , Humanos , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/toxicidade , Camundongos , Mycobacterium ulcerans , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria Ultravioleta
11.
Bull Exp Biol Med ; 166(6): 759-765, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31028588

RESUMO

Campylobacter genus bacteria causing campylobacteriasis are difficult to culture. This fact necessitates creation of special approaches to studies of the behavior of these pathogens during the manufacture and storage of foodstuffs. The regularities of Campylobacter jejuni transition into an uncultivable state are studied under conditions simulating the process of immersion cooling of fresh poultry products. The proportion of viable colony-forming (CFU) cells to the total count of planktonic and uncultivable cells in the population was calculated by the level of genomic DNA in the samples evaluated by quantitative real-time PCR with intercalating dyes. PCR was carried out with primers detecting the cytolethal toxin subunit B gene cdtB and invasion gene ciaB in C. jejuni strains. The count of detected cells was 5-10-fold higher than the count of CFU; the cultural method failed to detect the agent in 40% analyzed samples of superficially infected products, while the level of uncultivable cells detected by PCR was significantly higher. The relationship between culturing conditions and formation of C. jejuni biofilms was studied. The most intensive formation of film exomatrix was observed under unfavorable conditions for this microorganism at 25oC. In microaerophilic gaseous medium, weak formation of films and intensive growth of C. jejuni populations were observed. Culturing at higher temperatures (37-42oC) was in fact inessential for the film formation process.


Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Campylobacter jejuni/crescimento & desenvolvimento , Produtos Avícolas/microbiologia , Animais , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Carga Bacteriana , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Galinhas , Contagem de Colônia Microbiana , Contaminação de Alimentos/análise , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , Temperatura Ambiente
13.
Toxins (Basel) ; 11(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823654

RESUMO

Clostridium perfringens type E is a less frequently isolated C. perfringens type and has not previously been reported in France. We have characterized two recent type E isolates, C. perfringens 508.17 from the intestinal content of a calf that died of enterotoxemia, and 515.17 from the stool of a 60-year-old woman, subsequent to food poisoning, which contained the plasmid pCPPB-1 with variant iota toxin and C. perfringens enterotoxin genes.


Assuntos
Clostridium perfringens/isolamento & purificação , ADP Ribose Transferases/biossíntese , ADP Ribose Transferases/isolamento & purificação , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/isolamento & purificação , Bovinos , Sobrevivência Celular , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Enterotoxemia/microbiologia , Fezes/microbiologia , Feminino , França , Humanos , Intestinos/microbiologia , Pessoa de Meia-Idade , Filogenia , Células Vero
14.
Biosens Bioelectron ; 130: 73-80, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30731348

RESUMO

Current genetic detection methods require gene isolation, gene amplification and detection with a fluorescent-tagged probe. They typically require sophisticated equipment and expensive fluorescent probes, rendering them not widely available for rapid acute infection diagnoses at the point of care to ensure timely treatment of the diseases. Here we report a rapid genetic detection method that can detect the bacterial gene directly from patient stools using a piezoelectric plate sensor (PEPS) in conjunction with a continuous flow system with two temperature zones. With stools spiked with sodium dodecyl sulfate (SDS) in situ bacteria lysing and DNA denaturation occurred in the high-temperature zone whereas in situ specific detection of the denatured DNA by the PEPS occurred in the lower-temperature zone. The outcome was a rapid genetic detection method that directly detected bacterial genes from stool in < 40 min without the need of gene isolation, gene amplification, or expensive fluorescent tag but with polymerase chain reaction (PCR) sensitivity. In 40 blinded patient stools, it detected the toxin B gene of Clostridium difficile with 95% sensitivity and 95% specificity. The all-electrical, label-free nature of the detection further supports its potential as a low-cost genetic test that can be used at the point of care.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Técnicas Biossensoriais , Clostridium difficile/isolamento & purificação , Fezes/microbiologia , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridium difficile/genética , Clostridium difficile/patogenicidade , Humanos , Dodecilsulfato de Sódio
15.
Ecotoxicol Environ Saf ; 172: 488-503, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738231

RESUMO

Biological treatment of cyanotoxins has gained much importance in recent decades and holds a promise to work in coordination with various physicochemical treatments. In drinking water treatment plants (DWTPs), effective removal of cyanotoxins with reduced toxicity is a primary concern. Commonly used treatments, such as ozonation, chlorination or activated carbon, undergo significant changes in their operating conditions (mainly dosage) to counter the variation in different environmental parameters, such as pH, temperature, and high cyanotoxin concentration. Presence of metal ions, natural organic matter (NOM), and other chemicals demand higher dosage and hence affect the activation energy to efficiently break down the cyanotoxin molecule. Due to these higher dose requirements, the treatment leads to the formation of toxic metabolites at a concentration high enough to break the guideline values. Biological methods of cyanotoxin removal proceed via enzymatic pathway where the protein-encoding genes are often responsible for the compound breakdown into non-toxic metabolites. However, in contrast to the chemical treatment, the biological processes advance at a much slower kinetic rate, predominantly due to a longer onset period (high lag phase). In fact, more than 90% of the studies reported on the biological degradation of the cyanotoxins attribute the biodegradation to the bacterial suspension. This suspended growth limits the mass transfer kinetics due to the presence of metal ions, NOMs and, other oxidizable matter, which further prolongs the lag phase and makes biological process toxic-free, albeit less efficient. In this context, this review attempts to bring out the importance of the attached growth mechanism, in particular, the biofilm-based treatment approaches which can enhance the biodegradation rate.


Assuntos
Toxinas Bacterianas/isolamento & purificação , Água Potável/química , Poluentes Químicos da Água/isolamento & purificação , Purificação da Água , Biodegradação Ambiental , Biofilmes , Reatores Biológicos , Cianobactérias/metabolismo , Monitoramento Ambiental , Microcistinas/isolamento & purificação
16.
Biosens Bioelectron ; 128: 91-96, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30640125

RESUMO

This paper demonstrates the use of a smartphone-based sensor for fluorescence polarization (FP) analysis of biomolecules. The FP detection can rapidly sense ligand-analyte bindings by measuring molecule mobility, and thus, FP-based assays have been widely used for rapid diagnostics in clinics. Here, we implemented the FP detection apparatus using a 3D-printed compact holder and the built-in camera of a smartphone. The system offers accurate measurements of the degree of polarization by simultaneously detecting the fluorescence intensities parallel and perpendicular to the polarization of the excitation. The fluorescence signal of the sample is excited by a laser or light-emitting diode and separated by a polarization beam cube depending on the polarization. Parallel and perpendicular polarized emissions are projected onto two different regions of the sensor chip in the smartphone camera. A custom software app was developed to count the average intensity in the areas of interest and compute the degree of polarization. We validated the system by measuring the polarization of dye molecules dissolved in solutions with different viscosities. As an example of biomolecule sensing, a competitive FP immunoassay of Prostaglandin E2 was demonstrated using the developed system and exhibited the limit of detection of 1.57 ng/mL. The smartphone-based FP assay platform can also be implemented for the detection of toxins, disease biomarkers, and pathogens in resource-limited settings.


Assuntos
Toxinas Bacterianas/química , Biomarcadores/química , Técnicas Biossensoriais , Polarização de Fluorescência , Toxinas Bacterianas/isolamento & purificação , Humanos , Lasers , Ligantes , Limite de Detecção , Smartphone
17.
FEMS Microbiol Lett ; 366(2)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30596999

RESUMO

Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter.


Assuntos
Toxinas Bacterianas/toxicidade , Bombyx/efeitos dos fármacos , Proteínas Hemolisinas/toxicidade , Inseticidas/farmacologia , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Bombyx/microbiologia , Enterobacter/classificação , Enterobacter/genética , Enterobacter/isolamento & purificação , Enterobacter/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/isolamento & purificação , Inseticidas/química , Inseticidas/metabolismo , Filogenia , Domínios Proteicos
18.
J Microbiol Immunol Infect ; 52(2): 242-247, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30181097

RESUMO

BACKGROUND: Carriage of methicillin-resistant Staphylococcus aureus (MRSA) is associated with its transmission. International travels and massive gatherings may accelerate such transmission. MRSA carriage was surveyed among the attendees of two international medical conferences held in Taipei in 2010. METHODS: A total of 209 attendees from 23 countries were recruited. Nasal specimens were collected from each volunteer and subjected to polymerase chain reaction (PCR) detection for MRSA. Molecular analysis, including pulsed-field gel electrophoresis, multilocus sequence typing (MLST), typing of staphylococcal cassette chromosome mec (SCCmec) and staphylococcal protein A (spa) genes, and detection of Panton-Valentine leukocidin (PVL) and sasX genes, was performed. RESULTS: MRSA carriage was detected in 10 (4.8%) attendees from Vietnam (3/8, 37.5%), Korea (2/6, 33.3%), Japan (2/41, 4.9%), Philippines (2/52, 3.8%), and Bangladesh (1/4, 25.0%). The proportion of MRSA colonizers was significantly higher in the local hospital group compared to those from the other groups (3/17 vs. 7/192, p < 0.05). Six MRSA isolates were available for molecular analysis. They all carried a type IV SCCmec gene. Five pulsotypes were identified; four genotypes, respectively, were identified by MLST and spa typing. None of the isolates carried either PVL or sasX genes. None of common molecular characteristics was shared by isolates from different countries. Most of these isolates were local endemic community clone in each country. CONCLUSIONS: As healthcare workers, a certain proportion of international medical conference attendees harbored MRSA in their nares, mostly local endemic community clones in each country, which has the potential of spread among attendees.


Assuntos
Pessoal de Saúde , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Cavidade Nasal/microbiologia , Adulto , Toxinas Bacterianas/isolamento & purificação , Técnicas de Tipagem Bacteriana , Portador Sadio , Congressos como Assunto , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Exotoxinas/isolamento & purificação , Feminino , Genes Bacterianos/genética , Genótipo , Humanos , Leucocidinas/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem Molecular , Tipagem de Sequências Multilocus , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/transmissão , Proteína Estafilocócica A/genética , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação
19.
PLoS One ; 13(11): e0206815, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30403719

RESUMO

NetF-producing type A Clostridium perfringens is an important cause of canine and foal necrotizing enteritis. NetF, related to the ß-sheet pore-forming Leukocidin/Hemolysin superfamily, is considered a major virulence factor for this disease. The main purpose of this work is to demonstrate the pore-forming activity of NetF and characterize the chemical nature of its binding site. Electron microscopy using recombinant NetF (rNetF) confirmed that NetF is able to oligomerize and form large pores in equine ovarian (EO) cell membranes and sheep red blood cells. These oligomeric pores appear to be about 4-6 nm in diameter, and the number of oligomer subunits to vary from 6 to 9. Sodium periodate treatment rendered EO cells non-susceptible to NetF, suggesting that NetF binding requires cell surface carbohydrates. NetF cytotoxicity was also inhibited by a lectin that binds sialic acid, by sialidase, and by free sialic acid in excess, all of which clearly implicate sialic acid-containing membrane carbohydrates in NetF binding and/or toxicity for EO cells. Binding of NetF to sheep red blood cells was not inhibited by the gangliosides GM1, GM2 and GM3, nor did the latter promote membrane permeabilization in liposomes, suggesting that they do not constitute the cellular receptors. In contrast, treatment of EO cells with different proteases reduced their susceptibility to NetF, suggesting that the NetF receptor is a sialic acid-containing glycoprotein.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridium perfringens/patogenicidade , Enterite/patologia , Enterotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Animais , Toxinas Bacterianas/isolamento & purificação , Toxinas Bacterianas/toxicidade , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Clostridium perfringens/metabolismo , Enterite/microbiologia , Enterite/veterinária , Enterotoxinas/isolamento & purificação , Enterotoxinas/toxicidade , Eritrócitos , Feminino , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/ultraestrutura , Cavalos , Interações entre Hospedeiro e Microrganismos , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica , Ovário/citologia , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Proteínas Recombinantes/ultraestrutura , Ovinos
20.
PLoS One ; 13(11): e0206813, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30383811

RESUMO

Bacillus thuringiensis (Bt) constitutes the active ingredient of many successful bioinsecticides used in agriculture. In the present study, the genetic diversity and toxicity of Bt isolates was investigated by characterization of native isolates originating from soil, fig leaves and fruits from a Turkish collection. Among a total of 80 Bt isolates, 18 of them were found carrying a vip3 gene (in 23% of total), which were further selected. Insecticidal activity of spore/crystal mixtures and their supernatants showed that some of the Bt isolates had significantly more toxicity against some lepidopteran species than the HD1 reference strain. Five isolates were analyzed by LC-MS/MS to determine the Cry protein composition of their crystals. The results identified the Cry1Ac protein and a Cry2A-type protein in all isolates, Cry1Ea in 3 of them and Cry1Aa in one. The sequence analysis of the new vip3 genes showed that they had a high similarity to either vip3Aa, vip3Af or vip3Ag (94-100%). The vip3Aa gene of the 6A Bt isolate was cloned and sequenced. The protein was named Vip3Aa65 by the Bacillus thuringiensis Nomenclature Committee. The expressed and purified Vip3Aa65 protein was tested against five lepidopteran species and its toxicity compared to that of a reference protein (Vip3Aa16). Both proteins had similar toxicity against Grapholita molesta and Helicoverpa armigera, whereas Vip3Aa65 was less active than Vip3Aa16 against three species from the Spodoptera genus. A tetrameric structure of the Vip3Aa65 protein was detected by gel filtration chromatography. The study revealed some isolates with high insecticidal activity which can be considered promising candidates to be used in pest control.


Assuntos
Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Mariposas/microbiologia , Controle Biológico de Vetores/métodos , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Ficus/microbiologia , Frutas/microbiologia , Mariposas/patogenicidade , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Folhas de Planta/microbiologia , Microbiologia do Solo , Turquia
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