ABSTRACT
BACKGROUND & OBJECTIVES: Streptococcus agalactiae (group B streptococci, GBS) is one of the leading causative agents of human and animal infections. Recently it was demonstrated that integration of different IS elements could inactivate some of the GBS virulence properties. The presence of IS elements in human isolates has been studied while the bovine isolates were not investigated till now. The objective of the study was to perform IS analysis of a large number of bovine GBS and to use the IS elements for classification and molecular epidemiology of GBS strains. METHODS: A total of 101 GBS isolates obtained from the dairy cows were tested. These were analyzed by PCR and multiplex PCR. Southern hybridization was accomplished with the Enzo(TM) DNA Labeling and Detection Kit. The computer techniques were used for selection of the specific primers and for analysis of the sizes of PCR products. RESULTS: GBS isolates collected at three different dairy farms were studied for the presence of IS elements. Multiplex PCR was used for the fast screening. It was found that IS861 presented in 29 GBS isolates (28.7%), IS1548 in 9 (8.9%), ISSa4 in 48 (47.5%) and IS1381 in 26 isolates (25.7%). A total of 28 bovine GBS isolates (27.7%) did not possess any of the IS elements, 36 (35.6%) possessed, 35 (34.7%) possessed two and 2 (1.9%) possessed three different IS elements. The GBS with four different IS elements were not found. Taken together, 10 different variants of GBS strains were discovered. Two out of 10 variants being specific for 51 isolates (50.5%) were predominant in bovine GBS. The results of the study demonstrated that the presence of IS elements significantly varied in bovine GBS. INTERPRETATION & CONCLUSION: The present data demonstrated that variants of IS elements present in GBS genome could be used as effective criteria for molecular epidemiology. In future this approach could probably be used as an additional tool for the epidemiological control and prevention of other bacterial infections.
Subject(s)
Animals , Base Sequence , Cattle , DNA Primers , DNA Transposable Elements , Female , Polymerase Chain Reaction , Streptococcus agalactiae/genetics , Virulence/geneticsABSTRACT
BACKGROUND & OBJECTIVES: immunocompromized adults. Approximately 50 per cent of the GBS strains carry and express the gene of BAC antigen which is capable to bind IgA. Gene encoding for the BAC antigen has been cloned and sequenced but actual IgA binding region on the protein has not been detected. The aim of the present work was to localize the region of IgA binding on Bac protein, to evaluate the role of one of the Bac protein regions MLKKIE in IgA binding, and to investigate the ability of Bac based recombinant proteins to generate protective antibodies against GBS infection. METHODS: Recombinant proteins based on beta antigen C were generated after PCR amplification of the fractions of bac gene with the following cloning of the PCR products into expression plasmids. Recombinant peptides were tested for IgA binding by immunoprecipitation and Western blot. One of the recombinant proteins expressing IgA binding was used as an antigen for immunization of mice and for GBS protection studies. RESULTS: Several bac gene constructs were generated. Their ability to bind IgA varied dramatically depending on the size of the construct and location of the fragment on the bac gene map. The smallest peptide expressing IgA binding was 14 kD in size. Amino acid substitutions in MLKKIE region facilitated IgA binding ability. Immunization of mice with recombinant Bac based peptide induced the appearance of anti-GBS antibody with high affinity level providing protection against GBS infection. INTERPRETATION & CONCLUSION: Size dependence of Bac based recombinant peptides proved that the effective IgA binding required specific folding of the protein binding IgA. Region MLKKIE could not be considered as region, responsible for IgA binding. Generation of antibodies against Bac based recombinant peptides with high titre and affinity makes these proteins a potent candidates for generating a vaccine against GBS infection.
Subject(s)
Adult , Amino Acid Sequence , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/immunology , Bacterial Vaccines/immunology , Base Sequence , DNA Primers , Humans , Infant, Newborn , Mice , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Proteins/immunology , Streptococcal Infections/immunologyABSTRACT
BACKGROUND & OBJECTIVES: The goal of the present study was to improve and simplify the diagnosis of Streptococcus agalactiae (group B Streptococcus, GBS) infection for routine clinical practice. METHODS: A total of 71 clinical samples were tested by microbiologic culture, counter immunoelectrophoresis (CIE) and PCR described in the literature. Southern hybridization was accomplished with the Enzo(TM) "DNA Labeling and Detection Kit", Roche (Germany). The computer techniques were used for selection of the specific primers and for analysis of the sizes of PCR products. RESULTS: The primers for the regions around the 51 bp deletion in C5a peptidase gene (scpB) of GBS were selected. PCR analysis revealed the 255 bp amplification fragment in GBS, 306 bp fragment in groups A and G streptococci (GAS, GGS) and did not reveal any fragments in other bacterial species. Among 71 urine and serum clinical samples tested, none were found to be GBS positive by microbiologic culture, 16 samples by CIE, 36 by PCR. The specificity of amplification was confirmed by Southern hybridization. INTERPRETATION & CONCLUSION: The 51 bp deletion in scpB gene in comparison with scpA and scpG genes can be used as a diagnostic tool for identification of GBS. The 51 bp deletion based PCR proved to be faster and more reliable test than microbiologic culture or CIE.
Subject(s)
Adhesins, Bacterial/genetics , Base Sequence , DNA Primers , Endopeptidases/genetics , Female , Genes, Bacterial , Humans , Polymerase Chain Reaction/methods , Pregnancy , Streptococcal Infections/diagnosis , Streptococcus agalactiae/geneticsABSTRACT
El bacteriófago virulento C1 es capaz de reproducirse en estreptococos de los grupos C y A, bacterias capaces de causar enfermedades en animales y en el hombre. Un conocimiento mayor del fago C1 podría contribuir al estudio de los estreptococos correspondientes mediante el intercambio genético. Los resultados de este trabajo muestran que el ADN-C1 presenta una densidad de flotación de 1,705 g/cm a la tres-CsC1, temperaturas de fusión de 75 y 88-C. Siete de las 15 restrictasas incubadas con el ADN-C1 no exhibieron sitio de reconocimiento en su secuencia, el mapa físico fue construido para las ocho endonucleasas restantes. Fueron clonados los fragmentos B y B-C Hind III en pBR 322. Los estudios preliminares de estos clones no mostraron la presencia de LAF en los extractos bacterianos
Subject(s)
Bacteriophages , Crossing Over, Genetic , DNA , StreptococcusABSTRACT
El bacteriófago C1 específico para la cepa C4540 se ha utilizado para la producción de una lisozima asociada al fago (LAF), que tiene la propiedad de lisar estreptococos y otras bacterias. Sin embargo, poco se conoce de las características biomoleculares de este fago, que es capaz de reproducirse en otras cepas del grupo C de estreptococos, y del grupo A, bacterias capaces de producir enfermedades en animales y en el hombre. Mediante el intercambio genético entre el fago y los correspondientes estreptococos, podrían ser estudiados los factores genéticos de patogenicidad de estas bacterias, para lo cual se hace necesario el conocimiento de las características del propio bacteriofago. En el presente trabajo se estudiaron las características morfológicas, fisicoquímicas y biomoleculares de la partícula del fago C1 y sus proteínas. La microscopia electrónica muestra que el fago C1 posee una cabeza hexagonal de la cual surge un pequeño vástago constituido por 6-8 filamentos; la densidad de flotación de la partícula es de 1,462 g/cm a la 3 en CsC1; la cápsula del fago presenta siete polipéptidos electroforéticamente diferentes. Los datos evidenciaron la ausencia, en la estructura de la cápsula, de la potente lisozima asociada al fago C1, lo que contradice los dato de la literatura
Subject(s)
Bacteriophages , StreptococcusABSTRACT
Los estreptococos del grupo A son capaces de provocar enfermedades en el hombre. El estudio genético de los factores de patogenicidad de estas bacterias ha estado limitado a la transducción (por bacteriófagos), sin embargo, las complejas características de los correspondientes bacteriófagos no se han estudiado suficientemente. En el presente trabajo se investigaron las características fisicoquímicas de las partículas, proteínas y ADN de los bacteriófagos CA1 y A25 de estreptococos del grupo A. Los principales resultados mostraron que el fago CA1 es idéntico morfológicamente al A25, presentando una cabeza hexagonal (60 nm) con una larga cola no contráctil (180 nm), según los datos de la microscopía electrónica. La estructura de la partícula de ambos fagos está constituida por 10 polipéptidos electroforéticamente diferentes. Los ADNs de ambos fagos presentan características similares: densidad de flotación 1,709 g/cm a la tres CsCI; dos zonas de temperatura de fusión a 80- y 88-C; ambos fagos presentan moléculas de ADN lineales y de doble hebra de una longitud de 38,3ñ 1,1 kb. Los datos de los análisis de homología mostraron una complementariedad total entre los ADNs de estos fagos. La secuencia de los ADNs de los bateriófagos A25 y CA1 contiene sitios de reconocimiento para seis endonucleasas (Hae II, Msp I, Ben I, Hind III y Bsu RI); basado en los análisis de restricción se construyó el mapa físico preliminar para ambos ADNs