RESUMO
Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis.
Assuntos
Escherichia coli Enteropatogênica/genética , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/genética , Animais , Clonagem Molecular , Escherichia coli Enteropatogênica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Proteínas de Escherichia coli/biossíntese , Expressão Gênica , Sus scrofa/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/prevenção & controle , Ativação TranscricionalRESUMO
Patients undergoing neurosurgery are predisposed to a variety of complications related to mechanical ventilation (MV). There is an increased incidence of extubation failure, pneumonia, and prolonged MV among such patients. The aim of the present study was to assess the influence of extubation failure and prolonged MV on the following variables: postoperative pulmonary complications (PPC), mortality, reoperation, tracheostomy, and duration of postoperative hospitalization following elective intra-cranial surgery. The study involved a prospective observational cohort of 317 patients submitted to elective intracranial surgery for tumors, aneurysms and arteriovenous malformation. Preoperative assessment was performed and patients were followed up for the determination of extubation failure and prolonged MV (>48 h) until discharge from the hospital or death. The occurrence of PPC, incidence of death, the need for reoperation and tracheostomy, and the length of hospitalization were assessed during the postoperative period. Twenty-six patients (8.2 percent) experienced extubation failure and 30 (9.5 percent) needed prolonged MV after surgery. Multivariate analysis showed that extubation failure was significant for the occurrence of death (OR = 8.05 [1.88; 34.36]), PPC (OR = 11.18 [2.27; 55.02]) and tracheostomy (OR = 7.8 [1.12; 55.07]). Prolonged MV was significant only for the occurrence of PPC (OR = 4.87 [1.3; 18.18]). Elective intracranial surgery patients who experienced extubation failure or required prolonged MV had a higher incidence of PPC, reoperation and tracheostomy and required a longer period of time in the ICU. Level of consciousness and extubation failure were associated with death and PPC. Patients who required prolonged MV had a higher incidence of extubation failure.
Assuntos
Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Extubação/efeitos adversos , Encefalopatias/cirurgia , Malformações Arteriovenosas Intracranianas/cirurgia , Desmame do Respirador/efeitos adversos , Estudos de Coortes , Procedimentos Cirúrgicos Eletivos , Complicações Pós-Operatórias , Estudos Prospectivos , Respiração Artificial , Fatores de Risco , Fatores de TempoRESUMO
Patients undergoing neurosurgery are predisposed to a variety of complications related to mechanical ventilation (MV). There is an increased incidence of extubation failure, pneumonia, and prolonged MV among such patients. The aim of the present study was to assess the influence of extubation failure and prolonged MV on the following variables: postoperative pulmonary complications (PPC), mortality, reoperation, tracheostomy, and duration of postoperative hospitalization following elective intra-cranial surgery. The study involved a prospective observational cohort of 317 patients submitted to elective intracranial surgery for tumors, aneurysms and arteriovenous malformation. Preoperative assessment was performed and patients were followed up for the determination of extubation failure and prolonged MV (>48 h) until discharge from the hospital or death. The occurrence of PPC, incidence of death, the need for reoperation and tracheostomy, and the length of hospitalization were assessed during the postoperative period. Twenty-six patients (8.2%) experienced extubation failure and 30 (9.5%) needed prolonged MV after surgery. Multivariate analysis showed that extubation failure was significant for the occurrence of death (OR = 8.05 [1.88; 34.36]), PPC (OR = 11.18 [2.27; 55.02]) and tracheostomy (OR = 7.8 [1.12; 55.07]). Prolonged MV was significant only for the occurrence of PPC (OR = 4.87 [1.3; 18.18]). Elective intracranial surgery patients who experienced extubation failure or required prolonged MV had a higher incidence of PPC, reoperation and tracheostomy and required a longer period of time in the ICU. Level of consciousness and extubation failure were associated with death and PPC. Patients who required prolonged MV had a higher incidence of extubation failure.
Assuntos
Extubação/efeitos adversos , Encefalopatias/cirurgia , Malformações Arteriovenosas Intracranianas/cirurgia , Desmame do Respirador/efeitos adversos , Adulto , Estudos de Coortes , Procedimentos Cirúrgicos Eletivos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Estudos Prospectivos , Respiração Artificial , Fatores de Risco , Fatores de TempoRESUMO
AIMS: To assess the vital capacity (VC), tidal volume, minute volume and respiratory rate during the first four postoperative days of elective craniotomy and how they are correlated with smoking, associated diseases and respiratory symptoms. PATIENTS AND METHODS: Ninety-four patients were initially evaluated for elective craniotomy and they were included in this study only if they presented normal consciousness level and spontaneous breathing at the first postoperative. The preoperative and postoperative evaluations comprised physical examination and ventilometry up to the fourth postoperative. The repeated measures analysis of variance was used to the ventilation measurements. The significance level adopted for all the statistical tests was p = 0.05. RESULTS: Sixty-two patients were included in this study. There was a 20% fall in the VC from the first to the third postoperative (p = 0.001). Patients with systemic arterial hypertension presented in the preoperative period a lower mean VC (2.59 L) than the patients without (3.28 L) (p = 0.045). Smokers presented a lower mean VC (2.65 and 1.95 L) than the nonsmokers (3.13 and 2.43 L), both in the preoperative and in the postoperative, but with no statistic significance (p = 0.090). CONCLUSION: After elective craniotomy, there is a significant decrease in VC immediately after surgery, improving gradually thereafter; there was no difference in VC between the smoking and nonsmoking patients in the pre- and postoperative as well.
Assuntos
Craniotomia , Pulmão/fisiopatologia , Ventilação Pulmonar , Capacidade Vital , Adulto , Feminino , Humanos , Masculino , Período Pós-OperatórioRESUMO
Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.
Assuntos
Anaplasma marginale/genética , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Anaplasma marginale/isolamento & purificação , Anaplasma marginale/metabolismo , Anaplasmose/imunologia , Anaplasmose/microbiologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting , Brasil , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 microg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.
Assuntos
Antígenos de Protozoários/genética , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Toxoplasma/imunologia , Toxoplasma/metabolismoRESUMO
The purpose of this work was to evaluate protective activity against brain cyst formation in BALB/c mice intranasally vaccinated with recombinant proteins from Toxoplasma gondii. The recombinant proteins rROP2, rGRA5 and rGRA7 were used in vaccine preparation. Thirty-three female mice were divided into three groups, these animals received two doses by intranasal route at days 0 and 21 as follows; group 1 (G1, n=11) received 12.5 microg of each recombinant protein plus 0.5 microg of cholera toxin, group 2 (G2, n=11) received phosphate buffer saline (PBS) plus 0.5 microg of cholera toxin, and group 3 (G3, n=11) received PBS only. At challenge day (day 33) three animals from each group were euthanatized for IgA measure from intestine. Mice were infected orally with 50 cysts from the VEG strain at day 33. At challenge day the G1 animals had high immunoglobulin A levels, however, they only showed IgG antibody titers against rROP2 and rGRA7. Animals from G1 also exhibited strong resistance to cyst formation compared with the control group (G3, P<0.05). However, we did not observe differences in protection against brain cyst formation between G1 and G2 (P>0.1). These results indicate that intranasal immunization in BALB/c mice with recombinant proteins rROP2, rGRA5 and rGRA7 associated with cholera toxin induced partial protection, when compared with G3, against tissue cyst formation after oral infection with tissue cysts from T. gondii.
Assuntos
Proteínas de Protozoários/imunologia , Vacinas Protozoárias/normas , Toxoplasma/imunologia , Toxoplasmose Cerebral/prevenção & controle , Administração Intranasal , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Western Blotting , Encéfalo/parasitologia , Eletroforese em Gel de Poliacrilamida , Feminino , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Organismos Livres de Patógenos Específicos , Toxoplasmose Cerebral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/normasRESUMO
Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.
Assuntos
Animais , Anaplasma marginale/genética , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Anaplasma marginale/isolamento & purificação , Anaplasma marginale/metabolismo , Anaplasmose/imunologia , Anaplasmose/microbiologia , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Western Blotting , Brasil , Clonagem Molecular , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Análise de Sequência de DNARESUMO
Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.
Assuntos
Animais , Antígenos de Protozoários/genética , Proteínas de Membrana/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Toxoplasma/imunologia , Toxoplasma/metabolismoRESUMO
Extended-spectrum beta-Lactamase-producing (ESBL) Klebsiella sp.isolates from an outbreak in a Neonatal Intensive Care Unit (NICU) at a teaching hospital in Londrina, Paraná State, Brazil, presented atypical phenotypic characteristics that hampered their identification and the distinction between Klebsiella and Enterobacter species. Ten isolates were identified as K. pneumoniae due to negative reactions for motility and inducible beta-lactamase test (ESBL and AmpC) despite being positive for ornithyne descarboxilase. These isolates were genotyped by ribotyping and polymerase chain reaction (PCR) with repetitive extragenic palindromic sequences (REP). Ribotyping by means of an automated instrument and EcoRI and Pvu II as restriction enzymes resulted indetection of K. pneumoniae subspecie pneumoniae RIBO1 222-36-S-5 ribotype. Typing by REP-PCR showed that the 17 isolates from the outbreak were highly similar, belonging to one cluster with 100 percent of similarity, and that they presented more than 70 percent of similarity with K. pneumoniae ATCC 13883 and ATCC 10031, and 25 percent of similarity with E. aerogenes CDC 1680. In conclusion, the isolates of the outbreak were identified as Klebsiella pneumoniae, despite presenting ornithyne descarboxilase enzyme, which is an atypical characteristic of this Klebsiella species.
Isolados de Klebsiella sp. produtora de beta-lactamase de espectro estendido (ESBL), responsável por um surto na Unidade Neonatal de Terapia Intensiva (UNTI) do Hospital Universitário de Londrina, Paraná, Brasil apresentaram características fenotípicas atípicas que dificultaram sua identificação e a diferenciação entre as espécies Klebsiella pneumoniae e Enterobacter aerogenes. Dez isolados foram identificados como K. pneumoniae devido às reações negativas para motilidade e produção de enzimas beta-lactamases (ESBL e AmpC). Embora apresentassem teste positivo para ornitina descarboxilase. Estes isolados foram genotipados por ribotipagem e por reação em cadeia da polimerase (PCR) com oligonucleotídeos para "repetitive extragenic palindromic sequences" (REP). A ribotipagem com as enzimas de restrição EcoRI e Pvu II detectou o ribotipo de K. pneumoniae subespécie pneumoniae RIBO1 222-36-S-5. A técnica de REP-PCR mostrou que os isolados do surto foram similares, pertencentes a um grupo com 100 por cento de similaridade, e apresentaram mais de 70 por cento de similaridade com amostras padrão de K. pneumoniae (ATCC 13883 e 10031), e 25 por cento de similaridade com E. aerogenes CDC 1680. Concluindo, os isolados do surto da NICU mostraram se geneticamente relacionados e foram identificados como Klebsiella pneumoniae, embora apresentassem ornitina descarboxilase, característica atípica para esta espécie de Klebsiella.