ES2307351B2 - STAFFYLOCOCCUS AUREUS LIVED LIVED CEPA AS A VACCINE IN RUMINANT MASTITIS. - Google Patents
STAFFYLOCOCCUS AUREUS LIVED LIVED CEPA AS A VACCINE IN RUMINANT MASTITIS. Download PDFInfo
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- ES2307351B2 ES2307351B2 ES200500809A ES200500809A ES2307351B2 ES 2307351 B2 ES2307351 B2 ES 2307351B2 ES 200500809 A ES200500809 A ES 200500809A ES 200500809 A ES200500809 A ES 200500809A ES 2307351 B2 ES2307351 B2 ES 2307351B2
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- C12R2001/44—Staphylococcus
- C12R2001/445—Staphylococcus aureus
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Abstract
Cepa viva atenuada de Staphylococcus aureus como vacuna en mastitis de rumiantes. La presente invención proporciona una nueva cepa vacunal viva de S. aureus que, además de ser atenuada, se caracteriza por presentar como novedades más significativas unas mayores tasas de proliferación y persistencia en células epiteliales mamarias. Estas propiedades favorecen una respuesta inmune prolongada en el tiempo. La cepa se encuentra depositada y registrada en la Colección Española de Cultivos Tipo (CECT), Universidad de Valencia, Campus de Burjassot, Edificio de Investigación, 46100 Burjassot (Valencia), con la referencia CECT 7061. La estrategia utilizada en su construcción confiere una total estabilidad genética a la mutación. También se cumple la falta de marcadores de resistencia antibiótica en la cepa vacunal como requerimiento de seguridad biológica.Live attenuated strain of Staphylococcus aureus as a vaccine in ruminant mastitis. The present invention provides a new live vaccine strain of S. aureus that, in addition to being attenuated, is characterized by presenting as more significant novelties higher rates of proliferation and persistence in mammary epithelial cells. These properties favor a prolonged immune response over time. The strain is deposited and registered in the Spanish Type Culture Collection (CECT), University of Valencia, Burjassot Campus, Research Building, 46100 Burjassot (Valencia), with the reference CECT 7061. The strategy used in its construction confers a Total genetic stability to the mutation. The lack of antibiotic resistance markers in the vaccine strain as a biological safety requirement is also met.
Description
Cepa viva atenuada de Staphylococcus aureus como vacuna en mastitis de rumiantes.Live attenuated strain of Staphylococcus aureus as a vaccine in ruminant mastitis.
La presente invención se encuadra dentro del campo de la Sanidad Animal. De forma más concreta, la invención se refiere a la producción de una cepa viva de Staphylococcus aureus atenuada mediante la deleción de dos genes, genéticamente estable, sin marcadores de resistencia a antibióticos y con capacidad de proliferación y persistencia en células epiteliares mamarias. Constituye una nueva cepa vacunal para las mastitis de rumiantes.The present invention falls within the field of Animal Health. More specifically, the invention relates to the production of a live strain of Staphylococcus aureus attenuated by the deletion of two genetically stable genes, without markers of antibiotic resistance and with the ability to proliferate and persist in mammary epithelial cells. It constitutes a new vaccine strain for ruminant mastitis.
Staphylococcus aureus es una bacteria muy ubicua, habitante natural de la piel y mucosas de los mamíferos y causante de distintas enfermedades en el hombre y los 1 animales domésticos. En estos últimos, S. aureus está principalmente implicado en infecciones intramamarias, que ocasionan importantísimas pérdidas económicas en la producción bovina, ovina y caprina. Staphylococcus aureus is a very ubiquitous bacterium, a natural inhabitant of the skin and mucous membranes of mammals and the cause of various diseases in humans and the 1 domestic animals. In the latter, S. aureus is mainly involved in intramammary infections, which cause very important economic losses in bovine, sheep and goat production.
En los rumiantes S. aureus es el agente productor de mastitis más importante por su frecuencia y/o gravedad. En el ganado bovino se ha estimado que es responsable del 19 al 40% de las infecciones intramamarias, siendo gran parte de ellas subclínicas (Sutra and Poutrel, 1994, J Med Microbiol, 40: 79-89; Giraudo, et al., 1997, J Dairy Sci, 80: 845-53), en tanto que en los pequeños rumiantes, en los que también puede ocasionar infecciones intramamarias subclínicas, es la principal y más grave causa de mamitis clínicas. Los tratamientos con antibióticos formulados para las infecciones intramamarias generalmente fracasan en la eliminación de las infecciones por S. aureus, además incrementan las pérdidas en la producción lechera por los correspondientes periodos de supresión que se establecen en la comercialización de la leche. Desde hace muchos años el desarrollo de vacunas para controlar la mastitis producida por S. aureus en los rumiantes ha recibido una atención preferente y han sido muchos y muy variados los preparados vacunales ensayados: células vivas, bacterinas, toxoides, bacterinas-toxoides, paredes celulares aisladas, etc. (Foster T. J., 1991, Vaccine, 9: 221-7. Review); (Sutra and Poutrel, 1994, J Med Microbiol, 40: 79-89); (patente US4840794). La eficacia de la mayoría de las vacunas ensayadas, sin embargo, es bastante limitada. En ensayos de campo los mejores resultados, traducidos en una reducción de la frecuencia y la gravedad de las infecciones intramamarias pero no en una protección frente a nuevas infecciones, se han obtenido con vacunas que combinan un toxoide con bacterias muertas de cepas productoras de cápsula o pseudocápsula y diferentes adyuvantes (Watson, S.A., 1998 Int J Cancer.;75(6): 873-7.; Amorena, B., et al. 1994. Vaccine. 12(3):243-9.; Nordhaug et al., 1994, J. Dairy Sci.. 77(5): 1267-75.). También se han obtenido resultados inmunizando vacas (Nelson et al. 1991. Flem Vet J. 62: 111-125) y ratones (Mamo et al., 1994. FEMS Immunol. Med. Microbiol. 10(1): 47-53) con adhesinas de S. aureus. Últimamente se han desarrollado vacunas de DNA (patente CA2392756), sin embargo, aunque esperanzadoras, este tipo de vacunas presentan algunos problemas como son la necesidad de una gran cantidad de plásmido y su alto grado de pureza, lo que encarece y dificulta su producción (Dietrich, G. et al, 2003. Curr Opin Mol Ther. 5(1): 10-9. Review).In ruminants S. aureus is the most important mastitis producing agent due to its frequency and / or severity. In cattle, it has been estimated that it is responsible for 19% to 40% of intramammary infections, being a large part of them subclinical (Sutra and Poutrel, 1994, J Med Microbiol, 40: 79-89; Giraudo, et al ., 1997 , J Dairy Sci, 80: 845-53), while in small ruminants, which can also cause subclinical intramammary infections, it is the main and most serious cause of clinical mastitis. Antibiotic treatments formulated for intramammary infections generally fail to eliminate S. aureus infections, and increase losses in milk production due to the corresponding suppression periods established in the commercialization of milk. For many years the development of vaccines to control mastitis produced by S. aureus in ruminants has received preferential attention and the vaccine preparations tested have been many and varied: living cells, bacterins, toxoids, bacterin-toxoids, cell walls isolated, etc. (Foster TJ, 1991, Vaccine, 9: 221-7. Review); (Sutra and Poutrel, 1994, J Med Microbiol, 40: 79-89); (US4840794 patent). The efficacy of most of the vaccines tested, however, is quite limited. In field trials, the best results, translated into a reduction in the frequency and severity of intramammary infections but not in protection against new infections, have been obtained with vaccines that combine a toxoid with dead bacteria from capsule-producing strains or pseudocapsule and different adjuvants (Watson, SA, 1998 Int J Cancer.; 75 (6): 873-7 .; Amorena, B., et al . 1994. Vaccine. 12 (3): 243-9 .; Nordhaug et al. ., 1994, J. Dairy Sci. 77 (5): 1267-75.). Results have also been obtained by immunizing cows (Nelson et al . 1991. Flem Vet J. 62: 111-125) and mice (Mamo et al ., 1994. FEMS Immunol. Med. Microbiol. 10 (1): 47-53) with adhesins of S. aureus . Lately, DNA vaccines have been developed (patent CA2392756), however, although hopeful, these types of vaccines present some problems such as the need for a large amount of plasmid and its high degree of purity, which makes its production more difficult and difficult ( Dietrich, G. et al , 2003. Curr Opin Mol Ther. 5 (1): 10-9. Review).
En la última década se ha avanzado bastante en el conocimiento tanto de la compleja patogenia de la mamitis estafilocócica como de los mecanismos de defensa inmunes, específicos y no específicos, de la glándula mamaria. Se han asociado con la patogenicidad intramamaria de S. aureus distintos factores: componentes superficiales de la bacteria (adhesinas, proteína A, polisacáridos capsulares), toxinas, enzimas extracelulares y coagulasa (revisados por Sutra and Poutrel. J Med Microbiol. 1994. 40: 79-89). No obstante, se considera que tres factores principales podrían estar implicados en la virulencia de la bacteria: las adhesinas, los polisacáridos capsulares y las toxinas (especialmente la \alpha y la \beta).In the last decade, considerable progress has been made in the knowledge of both the complex pathogenesis of staphylococcal mamitis and the specific and non-specific immune defense mechanisms of the mammary gland. Different factors have been associated with the intramammary pathogenicity of S. aureus : surface components of the bacteria (adhesins, protein A, capsular polysaccharides), toxins, extracellular enzymes and coagulase (reviewed by Sutra and Poutrel. J Med Microbiol. 1994. 40: 79-89). However, it is considered that three main factors could be involved in the virulence of the bacterium: adhesins, capsular polysaccharides and toxins (especially α and β).
La \beta-toxina o \beta-hemolisina es una esfingomielinasa C dependiente de Mg^{2+} que degrada la esfingomielina presente en la capa externa de fosfolípidos de la membrana celular. La tisis celular que produce se correlaciona con el contenido de esfingomielina de la membrana, por ello, los eritrocitos de rumiantes, que tienen el porcentaje de esfingomielina más alto de todos los mamíferos, son los más sensibles. La mayoría de las cepas de S. aureus aisladas de infecciones intramamarias bovinas (75-100%) producen esta hemolisina (codificada por el gen hlb), mientras que las cepas humanas raramente la expresan (Aarestrup et al. 1999. APMIS. 107(4): 425-30; Larsen et al. 2002. Vet Microbiol. 85(1): 61-7.). Se ha sugerido que la \beta-toxina, al igual que otras esfingomielinasas bacterianas, podría contribuir a la patogénesis de S. aureus por su actividad sobre membranas celulares (incluida su participación en la ruptura del fagosoma). Sin embargo, la posible participación de la 13-toxina en la patogénesis de S. aureus, y en concreto en la infección mamaria, no se ha demostrado concluyentemente hasta la fecha. Así, en estudios con toxina purificada, se ha descrito que la \beta-toxina induce cambios inflamatorios moderados en la glándula mamaria de ratón (Calvinho et al. 1993. Zentralbl Veterinarmed B. 40(8): 559-68) y de conejo (Ward et al. 1979. J Comp Pathol. 89: 169-177) y que es citotóxica para células epiteliales mamarias bovinas en cultivo (aunque en menor medida que la a toxina), (Cifrian et al. 1996. Vet Microbiol. 48: 187-198). Por otra parte, los estudios con mutantes que no expresan la \beta-toxina son escasos y poco concluyentes (Bramley et al. 1989. Infect Immun. 57: 2489-2494; Cifrian et al. 1996. Vet Microbiol. 48: 187-198). En un modelo murino de artritis séptica, no se han encontrado diferencias en la virulencia entre cepas parentales y mutantes nulos del gen hlb (Nilsson et al. 1999. Infect Immun. 67: 1045-1049). Tampoco se ha descrito un papel claro de esta toxina en la virulencia en un modelo de infección corneal en conejos (O'Callaghan et al. 1997. Infect Immun. 65: 1571- 1578; Dajcs et al. 2002. DNA Cell Biol. 21: 375-382).The β-toxin or β-hemolysin is a Mg 2+ -dependent sphingomyelinase C that degrades sphingomyelin present in the outer phospholipid layer of the cell membrane. The cellular tisis that it produces correlates with the sphingomyelin content of the membrane, therefore, ruminant erythrocytes, which have the highest percentage of sphingomyelin of all mammals, are the most sensitive. Most S. aureus strains isolated from bovine intramammary infections (75-100%) produce this hemolysin (encoded by the hlb gene), while human strains rarely express it (Aarestrup et al . 1999. APMIS. 107 ( 4): 425-30; Larsen et al . 2002. Vet Microbiol. 85 (1): 61-7.). It has been suggested that β-toxin, like other bacterial sphingomyelinases, could contribute to the pathogenesis of S. aureus by its activity on cell membranes (including its participation in phagosome rupture). However, the possible involvement of 13-toxin in the pathogenesis of S. aureus , and specifically in breast infection, has not been conclusively proven to date. Thus, in studies with purified toxin, β-toxin has been described to induce moderate inflammatory changes in the mouse mammary gland (Calvinho et al . 1993. Zentralbl Veterinarmed B. 40 (8): 559-68) and rabbit (Ward et al . 1979. J Comp Pathol. 89: 169-177) and that it is cytotoxic for bovine mammary epithelial cells (although to a lesser extent than toxin), (Cifrian et al . 1996. Vet Microbiol. 48 : 187-198). On the other hand, studies with mutants that do not express β-toxin are scarce and inconclusive (Bramley et al . 1989. Infect Immun. 57: 2489-2494; Cifrian et al . 1996. Vet Microbiol. 48: 187- 198). In a murine model of septic arthritis, no differences in virulence were found between parental strains and null mutants of the hlb gene (Nilsson et al . 1999. Infect Immun. 67: 1045-1049). Nor has a clear role of this toxin in virulence been described in a model of corneal infection in rabbits (O'Callaghan et al . 1997. Infect Immun. 65: 1571-1578; Dajcs et al . 2002. DNA Cell Biol. 21 : 375-382).
S. aureus produce numerosas enzimas extracelulares como hialuronidasa, nucleasa, lipasa, catalasa, fosfatasa, estafiloquinasa y proteasas que también se han relacionado con la patogenicidad de la bacteria en las infecciones intramamarias (Sutra and Poutrel. J Med Microbiol. 1994. 40: 79-89), si bien, por el momento, el posible modo de acción patógena de estas enzimas se desconoce. S. aureus produces numerous extracellular enzymes such as hyaluronidase, nuclease, lipase, catalase, phosphatase, staphylokinase and proteases that have also been linked to the pathogenicity of the bacteria in intramammary infections (Sutra and Poutrel. J Med Microbiol. 1994. 40: 79 -89), although, at the moment, the possible mode of pathogenic action of these enzymes is unknown.
La catalasa es una enzima que descompone el peróxido de hidrógeno generado durante el metabolismo celular en agua y oxígeno molecular. Con frecuencia se ha sugerido que las catalasas bacterianas pueden jugar un papel relevante en la protección de los microorganismos frente a la acción bactericida derivada del metabolismo oxidativo de las células fagocíticas. Así, en diversos microorganismos, como Nocardia asteroides (Beaman et al. 1985. Infect Immun. 47: 135-141), Mycobacterium tuberculosis (Manca et al. 1999. Infect Immun. 67: 74-79), Candida albicans (Wysong et al. 1998. Infect Immun. 66: 1953-1961; Nakagawa et al. 2003. Microbiol Immunol. 47: 395-403), Campylobacter jejuni (Day et al. 2000. Infect Immun. 68: 6337-6345), Edwardsiella tarda (Mathew et al. 2001. Microbiology. 147: 449-457), y Helicobacter pylori (Basu et al. 2004. Helicobacter. 9: 211-216) se ha demostrado que la catalasa es un factor de virulencia puesto que influye en su capacidad para sobrevivir en el interior de células fagocíticas. En S. aureus no se ha determinado con precisión hasta la fecha el posible papel que la catalasa pueda jugar en su patogénesis, si es que lo juega. No obstante, desde hace más de 25 años y basándose en evidencias indirectas, varios autores relacionaron la actividad catalasa de S. aureus con su virulencia (Mandell, G., L. 1975. J Clin Invest. 55: 561-566; Kanafani and Martín. 1985. J Clin Microbiol. 21: 607-610; Nishihara et al. 1985. Microbiol Immunol. 29: 151-155; Martin and Chaven. 1987. Appl environ Microbiol. 53: 1207-1209). Sin embargo, en estudios en los que se ha comparado la patogenicidad de un mutante catalasa negativo con su cepa parental se ha comprobado que, aunque el mutante era más sensible al peróxido de hidrógeno in vitro, era igual de patógeno que su cepa parental en el modelo murino de abscesos cutáneos (Horsburgh et al. 2001b. Infect Immun. 69: 3744-3754). Resultados similares han sido descritos por Messina et al. (2002. FEBS Lett. 518: 107-110) quienes analizaron la virulencia de un mutante catalasa negativo de S. aureus en comparación con aislados clínicos de S. aureus catalasa positivos midiendo la capacidad de supervivencia de las bacterias en el hígado de ratones inoculados por vía intravenosa y concluyeron que la ausencia de actividad catalasa no disminuía la virulencia de S. aureus. El gen que codifica la catalasa (katA) en S. aureus ha sido secuenciado y caracterizado (Sanz et al. 2000. Microbiology. 146: 465-475).Catalase is an enzyme that breaks down hydrogen peroxide generated during cellular metabolism in water and molecular oxygen. It has often been suggested that bacterial catalases may play a relevant role in the protection of microorganisms against bactericidal action derived from the oxidative metabolism of phagocytic cells. Thus, in various microorganisms, such as Nocardia asteroides (Beaman et al . 1985. Infect Immun. 47: 135-141), Mycobacterium tuberculosis (Manca et al . 1999. Infect Immun. 67: 74-79), Candida albicans (Wysong et al . 1998. Infect Immun. 66: 1953-1961; Nakagawa et al . 2003. Microbiol Immunol. 47: 395-403), Campylobacter jejuni (Day et al . 2000. Infect Immun. 68: 6337-6345), Edwardsiella takes (Mathew et al . 2001. Microbiology. 147: 449-457), and Helicobacter pylori (Basu et al . 2004. Helicobacter. 9: 211-216) it has been shown that catalase is a virulence factor since it influences its ability to survive inside phagocytic cells. In S. aureus, the possible role that catalase can play in its pathogenesis, if it plays it, has not been accurately determined to date. However, for more than 25 years and based on indirect evidence, several authors related the Catalan activity of S. aureus with its virulence (Mandell, G., L. 1975. J Clin Invest. 55: 561-566; Kanafani and Martín, 1985. J Clin Microbiol. 21: 607-610; Nishihara et al . 1985. Microbiol Immunol. 29: 151-155; Martin and Chaven. 1987. Appl environ Microbiol. 53: 1207-1209). However, in studies in which the pathogenicity of a negative catalase mutant has been compared with its parental strain it has been found that, although the mutant was more sensitive to hydrogen peroxide in vitro , it was as pathogenic as its parental strain in the murine model of skin abscesses (Horsburgh et al . 2001b. Infect Immun. 69: 3744-3754). Similar results have been described by Messina et al . (2002. FEBS Lett. 518: 107-110) who analyzed the virulence of a negative catalase mutant of S. aureus compared to clinical isolates of S. aureus catalase positive by measuring the survival capacity of bacteria in the liver of inoculated mice intravenously and concluded that the absence of catalase activity did not decrease the virulence of S. aureus . The gene encoding catalase ( katA ) in S. aureus has been sequenced and characterized (Sanz et al . 2000. Microbiology. 146: 465-475).
Aunque S. aureus ha sido tradicionalmente considerado como un patógeno extracelular, diversos estudios in vivo e in vitro han demostrado que es capaz de adherirse y ser internalizado por una gran variedad de tipos celulares, pudiendo sobrevivir y, en ocasiones, multiplicarse en el interior de células epiteliales (Almeida et al. 1996. J. Dairy Sci. 79: 1021-1026; Bayles et al. 1998. Infect Immun. 66: 336-342; Kahl et al. 2000. Infect Immun. 68: 5385-5392; Brouillette et al. 2003. Microb Pathog. 35: 159-168; Hess et al. 2003. J Surg Res. 114: 42-49), endoteliales (Hamill et al. 1986. Infect Immun. 54: 833-836; Yao et al. 1995. Infect Immun. 63: 1835-1839; Menzies and Kourteva. 1998. Infect Immun. 66: 5994-5998), fibroblastos (Fowler et al. 2000. Eur J Cell Biol. 79: 672-679), osteoblastos (Hudson et al. 1995. Microb Pathog. 19: 409-419; Ahmed et al. 2001. Infect Immun. 69: 2872-2877) e, incluso, células fagocíticas (Gresham et al. 2000. J Immunol. 164: 3713-3722; Hébert et al. 2000. FEMS Microbiol Lett. 193: 57-62; Brouillette et al. 2003. Microb Pathog. 35: 159-168). La internalización de S. aureus por las células del hospedador puede contribuir a la persistencia de la infección al proporcionar una resistencia frente al sistema inmune y frente a los tratamientos antibióticos (Ferens and Bohach. 2000. J Lab Clin Med. 135: 225-230).Although S. aureus has traditionally been considered as an extracellular pathogen, several in vivo and in vitro studies have shown that it is capable of adhering and being internalized by a wide variety of cell types, being able to survive and, sometimes, multiply inside epithelial cells (Almeida et al . 1996. J. Dairy Sci. 79: 1021-1026; Bayles et al . 1998. Infect Immun. 66: 336-342; Kahl et al . 2000. Infect Immun. 68: 5385-5392; Brouillette et al . 2003. Microb Pathog. 35: 159-168; Hess et al . 2003. J Surg Res. 114: 42-49), endothelial (Hamill et al . 1986. Infect Immun. 54: 833-836; Yao et al . 1995. Infect Immun. 63: 1835-1839; Menzies and Kourteva. 1998. Infect Immun. 66: 5994-5998), fibroblasts (Fowler et al . 2000. Eur J Cell Biol. 79: 672-679), osteoblasts (Hudson et al . 1995. Microb Pathog. 19: 409-419; Ahmed et al . 2001. Infect Immun. 69: 2872-2877) and even phagocytic cells (Gresham et al . 2000. J Immunol. 164: 3713-3722; Hébert e t al . 2000. FEMS Microbiol Lett. 193: 57-62; Brouillette et al . 2003. Microb Pathog. 35: 159-168). Internalization of S. aureus by host cells can contribute to the persistence of infection by providing resistance against the immune system and antibiotic treatments (Ferens and Bohach. 2000. J Lab Clin Med. 135: 225-230 ).
La invención consiste en la construcción de una cepa viva atenuada, de S. aureus, para ser utilizada como vacuna, incapaz de producir actividad catalasa y \beta-toxina, genéticamente estable y sin marcadores de resistencia antibiótica. Se considera cepa viva atenuada aquella estirpe que mantiene su capacidad de invadir y multiplicarse en el interior de su hospedador, pero sin desarrollar el cuadro patogénico.The invention consists in the construction of a live attenuated strain, of S. aureus , to be used as a vaccine, incapable of producing catalase and β-toxin activity, genetically stable and without antibiotic resistance markers. Live strain is considered to be that strain that maintains its ability to invade and multiply inside its host, but without developing the pathogenic picture.
Aunque la información publicada no permitía aventurar el uso de los genes katA y hlb como dianas para construir una cepa viva atenuada de S. aureus con aplicación vacunal, la construcción del doble mutante por deleción de estos dos genes ha supuesto la obtención de una nueva cepa vacunal. Se ha elegido una estrategia basada en la deleción en fase de la totalidad de los genes que codifican la catalasa, katA y la \beta-toxina, hlb, mediante doble recombinación homóloga.Although the published information did not allow to venture the use of the katA and hlb genes as targets to build a live attenuated strain of S. aureus with vaccination application, the construction of the double mutant by deletion of these two genes has meant obtaining a new strain vaccination A strategy based on the phase deletion of all the genes encoding catalase, katA and β-toxin, hlb , by double homologous recombination has been chosen.
El método utilizado para realizar esta invención cumple los requerimientos indispensables de seguridad biológica en cuanto a vacunas de nueva generación. Por un lado, asegura la estabilidad de la mutación puesto que la reversión al fenotipo silvestre está totalmente impedida debido a la ausencia de dianas requeridas para la recombinación homóloga o ilegítima a partir de poblaciones naturales de bacterias. Por otro lado, la cepa construida carece de marcadores de resistencia a antibióticos.The method used to perform this invention meets the essential requirements of biological safety in As for new generation vaccines. On the one hand, it ensures the stability of the mutation since the reversion to the phenotype wild is totally prevented due to the absence of targets required for homologous or illegitimate recombination from natural populations of bacteria. On the other hand, the strain Built lacks antibiotic resistance markers.
A pesar de los resultados conocidos hasta el momento, sorprendentemente, la novedosa combinación de las dos deleciones de los genes katA y hlb proporciona a esta cepa una dosis letal cincuenta (DL_{50}) más de 5 veces superior a la de la cepa parental (véase figura 3), siendo esta diferencia estadísticamente significativa. La avirulencia de esta cepa evidencia su empleo como cepa vacunal.In spite of the results known so far, surprisingly, the novel combination of the two deletions of the katA and hlb genes gives this strain a lethal dose fifty (DL 50) more than 5 times higher than that of the parental strain (see figure 3), this difference being statistically significant. The avirulence of this strain demonstrates its use as a vaccine strain.
La doble mutación tiene un claro efecto incrementando la proliferación y la persistencia del mutante en la línea celular MAC-T de células epiteliales mamarias y la persistencia en la línea celular de macrófagos J774A.1, observándose un efecto sinérgico y potenciador entre ambas mutaciones (véanse figuras 4 y 5). La proliferación del mutante es más lenta que la de la cepa parental, alcanzándose más tarde el máximo de bacterias intracelulares viables. La mayor permisividad para la replicación del mutante en células epiteliales mamarias es una consecuencia de su menor toxicidad para este tipo celular.The double mutation has a clear effect. increasing the proliferation and persistence of the mutant in the MAC-T mammary epithelial cell line and persistence in the J774A.1 macrophage cell line, observing a synergistic and potentiating effect between the two mutations (see figures 4 and 5). The proliferation of the mutant is slower than that of the parental strain, later reaching the maximum viable intracellular bacteria. The highest permissiveness for the replication of the mutant in mammary epithelial cells is a consequence of its lower toxicity for this cell type.
Los mayores niveles de proliferación y persistencia de la cepa atenuada en células epiteliales mamarias unidos a una mayor persistencia en células del sistema inmune (macrófagos) con respecto a su cepa parental, junto con la mayor DL_{50} en el modelo murino de infección, hacen del doble mutante una vacuna viva atenuada frente a las infecciones producidas por S. aureus y más concretamente frente a las mastitis producidas por este microorganismo.The higher levels of proliferation and persistence of the attenuated strain in mammary epithelial cells linked to a greater persistence in cells of the immune system (macrophages) with respect to their parental strain, together with the greater DL 50 in the murine model of infection, they make the double mutant a live attenuated vaccine against infections caused by S. aureus and more specifically against the mastitis produced by this microorganism.
Por lo tanto, un aspecto de esta invención incluye un kit de vacunación para mamíferos contra las enfermedades infecciosas producidas por S. aureus, que comprende los elementos y adyuvantes necesarios para contener y vehicular la cepa vacunal.Therefore, one aspect of this invention includes a vaccination kit for mammals against infectious diseases caused by S. aureus , which comprises the elements and adjuvants necessary to contain and vehicular the vaccine strain.
Otra aplicación de esta invención consiste en la utilización de la cepa atenuada como candidato ideal para ser utilizado como vector vacunal de expresión o transporte de antígenos heterólogos favorecido por su mayor persistencia en células del sistema inmune junto a su menor toxicidad y su mayor persistencia en el interior de células epiteliares mamarias con respecto a las cepas silvestres de S. aureus.Another application of this invention consists in the use of the attenuated strain as an ideal candidate to be used as a vaccine vector for the expression or transport of heterologous antigens favored by its greater persistence in cells of the immune system together with its lower toxicity and its greater persistence in the inside mammary epithelial cells with respect to the wild strains of S. aureus .
Para facilitar la comprensión de las principales características de la invención y formando parte integrante de esta memoria descriptiva, se acompañan una serie de figuras. Con carácter ilustrativo y no limitativo se ha representado lo siguiente:To facilitate the understanding of the main characteristics of the invention and forming an integral part of this Descriptive report, a series of figures are accompanied. With illustrative and non-limiting nature has been represented next:
Figura 1. Muestra el esquema de la construcción del plásmido termosensible pERhlb. Los oligonucleótidos utilizados en este estudio para la amplificación de fragmentos de ADN fueron diseñados a partir de secuencias del genoma de S. aureus COL (TIGR Microbial Database). Los números 1, 2, 3 y 4 se corresponden con la nomenclatura de los oligonucleótidos descritos en SEQ ID NO: 1, 2, 3 y 4, respectivamente.Figure 1. Shows the construction scheme of the thermosensitive plasmid pERhlb. The oligonucleotides used in this study for the amplification of DNA fragments were designed from sequences of the genome of S. aureus COL (TIGR Microbial Database). The numbers 1, 2, 3 and 4 correspond to the nomenclature of the oligonucleotides described in SEQ ID NO: 1, 2, 3 and 4, respectively.
Figura 2. Esquema de la construcción del plásmido termosensible pERkat. Los números 5, 6, 7 y 8 se corresponden con la nomenclatura de los oligonucleótidos descritos en SEQ ID NO: 5, 6, 7 y 8, respectivamente.Figure 2. Scheme of the construction of the thermosensitive plasmid pERkat. The numbers 5, 6, 7 and 8 are correspond to the nomenclature of the oligonucleotides described in SEQ ID NO: 5, 6, 7 and 8, respectively.
Figura 3. Ensayos de DL_{50} en el modelo murino. Se representan las medias aritméticas del número de ufc necesario para producir la muerte en el 50% de los animales de estudio más/menos la desviación estándar (DS). El valor de p obtenido en el análisis de varianza fue p=0,0032. Los valores representados son: (1) para S. aureus 2386 (2,80E+9) \pm DS 2,33E+9; (2) para su doble mutante katA/hlb, S. aureus CECT 7061, (1,41E+10) \pm DS 4,68E+9; y (3) para dicho mutante complementado con el plásmido que contiene ambos genes 5,77E+8 \pm DS 3,11E+8.Figure 3. DL 50 assays in the murine model. The arithmetic means of the number of cfu necessary to produce death in 50% of the study animals plus / minus the standard deviation (SD) are represented. The p value obtained in the analysis of variance was p = 0.0032. The values represented are: (1) S. aureus 2386 (2,80E + 9) \ ± SD 2,33E + 9; (2) for its double mutant katA / hlb , S. aureus CECT 7061, (1.41E + 10) ± SD 4.68E + 9; and (3) for said mutant supplemented with the plasmid containing both 5.77E + 8 ± DS 3.11E + 8 genes.
Figura 4. Supervivencia intracelular de S. aureus 2386, su doble mutante katA/hlb (S. aureus CECT 7061) y dicho mutante complementado con el plásmido que contiene ambos genes, en la línea celular macrofágica J774A.1 durante 24 horas. Con un rombo se representa la cepa 2386, con un cuadrado el doble mutante y mediante un triángulo el doble mutante complementado. En el eje de las ordenadas se representa el porcentaje (%) de bacterias intracelulares viables. Este dato se corresponde con la media del número de bacterias viables, más-menos su desviación estándar (\pmDS), expresado en porcentaje referido al total de bacterias presentes en el medio intracelular en el tiempo cero. En el eje de abscisas se señala el tiempo (T) en horas (h).Figure 4. Intracellular survival of S. aureus 2386, its double katA / hlb mutant ( S. aureus CECT 7061) and said mutant supplemented with the plasmid containing both genes, in the macrophage cell line J774A.1 for 24 hours. The strain 2386 is represented with a rhombus, with a square the double mutant and by a triangle the double mutant complemented. The percentage (%) of viable intracellular bacteria is represented on the ordinate axis. This data corresponds to the average number of viable bacteria, plus or minus their standard deviation (± SD), expressed as a percentage based on the total number of bacteria present in the intracellular medium at time zero. The time (T) in hours (h) is indicated on the abscissa axis.
Figura 5. Supervivencia intracelular de S. aureus 2386, su doble mutante katA/hlb (S. aureus CECT 7061) y dicho mutante complementado con el plásmido que contiene ambos genes, en la línea celular epitelial mamaria MAC-T. En los ejes de abscisa y ordenada se representan las mismas variables que en la figura 4. Asimismo, las diferentes cepas están señaladas por los mismos símbolos que en la figura anterior.Figure 5. Intracellular survival of S. aureus 2386, its double katA / hlb mutant ( S. aureus CECT 7061) and said mutant supplemented with the plasmid containing both genes, in the MAC-T mammary epithelial cell line. In the abscissa and ordinate axes the same variables are represented as in figure 4. Likewise, the different strains are indicated by the same symbols as in the previous figure.
Las cepas de S. aureus y E. coli empleadas se cultivaron rutinariamente a 37ºC con agitación en medio BHI (Infusión Cerebro-Corazón) y LB (Luria-Bertani), respectivamente. Las cepas transformadas con plásmidos termosensibles se incubaron a 32ºC para evitar la pérdida del plásmido. Los medios se complementaron con eritromicina (5 \mug/ml) cuando fue necesario. Para obtener medio sólido se añadió agar (15 g/l).The S. aureus and E. coli strains used were routinely grown at 37 ° C with shaking in BHI (Brain-Heart Infusion) and LB (Luria-Bertani), respectively. Strains transformed with heat sensitive plasmids were incubated at 32 ° C to prevent loss of the plasmid. The media were supplemented with erythromycin (5 µg / ml) when necessary. To obtain solid medium agar (15 g / l) was added.
Se utilizó S. aureus 2386 como cepa silvestre para construir las cepas mutantes Por otro lado, en los ensayos realizados con S. aureus 2386 y sus mutantes se utilizó un análisis de varianza. Cuando p fue menor de 0,05 (diferencias estadísticamente significativas) se realizó el test de Tukey-Kramer para un nivel de confianza del 95%. S. aureus 2386 was used as a wild strain to construct the mutant strains. On the other hand, in the tests performed with S. aureus 2386 and its mutants an analysis of variance was used. When p was less than 0.05 (statistically significant differences), the Tukey-Kramer test was performed for a 95% confidence level.
Habiendo descrito la presente invención, se ilustra adicionalmente mediante los siguientes ejemplos, los cuales no son limitativos de su alcance, que viene definido exclusivamente por la nota reivindicatoria adjunta.Having described the present invention, further illustrated by the following examples, which They are not limited to their scope, which is defined exclusively by the attached claim note.
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Construcción de un mutante por deleción del gen hlb, un mutante por deleción del gen katA y un doble mutante por deleción de ambos genes katA/hlb de S. aureus. Utilizando las parejas de oligonucleótidos descritos en SEQ ID NO: 1 y 2 por un lado y SEQ ID NO: 3 y 4 por otro se amplificaron por PCR dos fragmentos situados delante y detrás, respectivamente, del gen hlb (véase figura 1). Los fragmentos obtenidos se unieron mediante PCR recombinante siguiendo la técnica descrita por Vallejo et al. (1994) y se clonaron en pE194t, un plásmido derivado de S. aureus que confiere resistencia a la eritromicina, obteniendo el plásmido pERhlb. Este plásmido recombinante se introdujo en S. aureus 2386 mediante transformación de protoplastos, empleando el método descrito por Götz et al. (1981. J Bacteriol. 145: 74-81). Las bacterias transformadas, verificadas por PCR, se incubaron a 32ºC en presencia de eritromicina hasta la fase estacionaria de crecimiento. A continuación se elevó la temperatura a 43ºC durante 6 horas para inducir la recombinación homóloga entre el plásmido y el cromosoma bacteriano. Posteriormente se realizaron subcultivos a 37ºC sin eritromicina para favorecer la pérdida del plásmido. Los mutantes por deleción de hlb se seleccionaron en agar Columbia con un 5% de sangre de cordero por carecer de halo de b-hemólisis y ser CAMP negativos en presencia de Rhodococcus equi (Brzin, et al. 1990. Zentralbl Bakteriol. 273(2): 179-83). Por último se confirmó la deleción del gen hlb mediante técnicas de PCR.Construction of a mutant by deletion of the hlb gene, a mutant by deletion of the katA gene and a double mutant by deletion of both katA / hlb genes of S. aureus . Using the pairs of oligonucleotides described in SEQ ID NO: 1 and 2 on the one hand and SEQ ID NO: 3 and 4 on the other, two fragments located in front and behind, respectively, of the hlb gene were amplified by PCR (see Figure 1). The fragments obtained were joined by recombinant PCR following the technique described by Vallejo et al . (1994) and were cloned into pE194t, a plasmid derived from S. aureus that confers resistance to erythromycin, obtaining plasmid pERhlb. This recombinant plasmid was introduced into S. aureus 2386 by transformation of protoplasts, using the method described by Götz et al . (1981. J Bacteriol. 145: 74-81). The transformed bacteria, verified by PCR, were incubated at 32 ° C in the presence of erythromycin until the stationary phase of growth. The temperature was then raised to 43 ° C for 6 hours to induce homologous recombination between the plasmid and the bacterial chromosome. Subsequently, subcultures were performed at 37 ° C without erythromycin to favor the loss of the plasmid. Hlb deletion mutants were selected on Columbia agar with 5% lamb blood because they lacked b-hemolysis halo and were CAMP negative in the presence of Rhodococcus equi (Brzin, et al . 1990. Zentralbl Bakteriol. 273 (2 ): 179-83). Finally, deletion of the hlb gene was confirmed by PCR techniques.
El mutante por deleción de katA se construyó de la misma forma descrita anteriormente. Para ello, se utilizaron las parejas de oligonucleótidos descritos en SEQ ID NO: 5 y 6 por un lado y SEQ ID NO: 7 y 8 por otro para amplificar los fragmentos situados delante y detrás del gen katA, respectivamente (véase figura 2). Con el plásmido resultante, pERkat, se transformó la cepa silvestre S. aureus 2386 obteniéndose mutantes por deleción de katA. Los mutantes por deleción de katA se seleccionaron en medio sólido por la ausencia de actividad catalasa al ser puestos en contacto con peróxido de hidrógeno al 3%. Asimismo se comprobó la deleción del gen katA por PCR.The katA deletion mutant was constructed in the same manner described above. For this, the pairs of oligonucleotides described in SEQ ID NO: 5 and 6 on the one hand and SEQ ID NO: 7 and 8 on the other were used to amplify the fragments located in front of and behind the katA gene, respectively (see figure 2). With the resulting plasmid, pERkat, the wild strain S. aureus 2386 was transformed, obtaining mutants by deletion of katA . The katA deletion mutants were selected in solid medium due to the absence of catalase activity when contacted with 3% hydrogen peroxide. The deletion of the katA gene was also checked by PCR.
El doble mutante por deleción de hlb/katA se construyó transformando con el plásmido pERkat el mutante por deleción de hlb ya construido y siguiendo la misma estrategia descrita para el mutante por deleción de katA. Este doble mutante se encuentra depositado y registrado en la Colección Española de Cultivos Tipo (CECT), Universidad de Valencia, Campus de Burjassot, Edificio de Investigación, 46100 Burjassot (Valencia), con la referencia CECT 7061.The double mutant by deletion of hlb / katA was constructed by transforming with the plasmid pERkat the mutant by deletion of hlb already constructed and following the same strategy described for the mutant by deletion of katA . This double mutant is deposited and registered in the Spanish Type Culture Collection (CECT), University of Valencia, Burjassot Campus, Research Building, 46100 Burjassot (Valencia), with the reference CECT 7061.
Por último, todos los mutantes se complementaron utilizando el plásmido pHpS9, en el que se clonaron los genes hlb (pHhlb), katA (pHkat) y ambos (pHkathlb). En todos los casos, la complementación reprodujo en los mutantes el fenotipo de las cepa silvestre de la que derivaban, por lo que el fenotipo observado se asociaba a la deleción específica del gen.Finally, all mutants were complemented using plasmid pHpS9, in which the hlb (pHhlb), katA (pHkat) and both (pHkathlb) genes were cloned. In all cases, the complementation reproduced in the mutants the phenotype of the wild strain from which they were derived, so the observed phenotype was associated with the specific deletion of the gene.
Ensayos de supervivencia intracelular en las líneas celulares J774A.1 y MAC-T. Para estudiar si la pérdida de actividad catalasa, la falta de producción de b-toxina o la ausencia de las dos actividades afectan a la supervivencia y a la proliferación intracelular de S. aureus, se realizaron estudios de supervivencia intracelular en dos líneas celulares diferentes: macrófagos J774A.1 y células epiteliares mamarias MAC-T. Los macrófagos constituyen una de las piezas esenciales de la inmunidad innata, y participan activamente en la presentación de antígenos para inducir una inmunidad adquirida satisfactoria. Las células epiteliares mamarias se utilizaron como modelo de estudio in vitro del comportamiento de S. aureus en la glándula mamaria de los rumiantes.Intracellular survival assays in J774A.1 and MAC-T cell lines. To study whether the loss of catalase activity, the lack of b-toxin production or the absence of the two activities affect the survival and intracellular proliferation of S. aureus , intracellular survival studies were conducted on two different cell lines: macrophages J774A.1 and MAC-T mammary epithelial cells. Macrophages constitute one of the essential pieces of innate immunity, and actively participate in the presentation of antigens to induce satisfactory acquired immunity. Mammary epithelial cells were used as an in vitro study model of the behavior of S. aureus in the mammary gland of ruminants.
Las líneas celulares J774A.1 y MAC-T se mantuvieron con Dulbecco's modified Eagle medium (DMEM) al que se añadió un 10% de suero fetal bovino (FBS) y un 1% de Pen/Strep/Fungizones Mix (Biowhittaker) y las incubaciones se hicieron a 37ºC con un 5% de CO_{2}. El medio de crecimiento de la línea MAC-T contenía además 5 \mug de insulina/ml y 1 \mug de hidrocortisona/ml. S. aureus 2386, sus mutantes por deleción de katA, hlb y katA/hlb, así como los tres mutantes complementados, se cultivaron durante 18-20 horas en medio BHI a 37ºC con agitación. Los cultivos se centrifugaron, se lavaron dos veces con tampón fosfato salino (PBS) pH 7, se resuspendieron en PBS + 20% Glicerol y se conservaron a -80ºC en alícuotas de 1 ml. Se determinó la concentración bacteriana de las alícuotas tras su congelación sembrando diluciones seriadas en placas de BHI. Para preparar las suspensiones bacterianas se descongelaron las alícuotas necesarias, se lavaron dos veces con PBS pH 7 y se resuspendieron a la concentración deseada (3-5 x 105 unidades formadoras de colonias/ml (ufc/ml)) en medio de invasión (medio de cultivo celular sin antibióticos). Los ensayos de supervivencia intracelular se realizaron en placas de 24 pocillos. Se inocularon los pocillos con 3 x 10^{4} células y se incubaron durante 48 horas en las condiciones descritas anteriormente. Aproximadamente 16 horas antes del ensayo se lavaron las células con PBS estéril y se reemplazó el medio de cultivo por medio de invasión. Antes del experimento se contaron las células presentes en uno de los pocillos para comprobar que hubiera 5-6 x 10^{4} células/pocillo. Para el experimento se añadió nuevo medio de invasión a las células y se inocularon los pocillos con las suspensiones bacterianas a una multiplicidad de infección de 10:1 (J774A.1) ó 50:1 (MAC-T). A continuación, las placas se centrifugaron a 1500 rpm durante 5 minutos y se incubaron durante 1 hora a 37ºC con 5% de CO_{2}. Tras esta incubación, se retiró el medio, se lavaron las células dos veces con PBS estéril y se añadió medio de invasión con 100 \mug de gentamicina/ml a cada pocillo para destruir las bacterias extracelulares. Después de la incubación con gentamicina durante 1 (bacterias internalizadas), 4, 8 y 24 horas, se realizaron dos lavados con PBS estéril y se lisaron las células infectadas añadiendo Tritón x-100 al 0,2%. Se hicieron diluciones seriadas de los lisados y se sembraron en placas de BHI para hacer el recuento de ufc. Las bacterias intracelulares supervivientes a las 4, 8 y 24 horas se expresaron como porcentaje de la bacterias intracelulares presentes en la primera hora.The J774A.1 and MAC-T cell lines were maintained with Dulbecco's modified Eagle medium (DMEM) to which 10% fetal bovine serum (FBS) and 1% Pen / Strep / Fungizones Mix (Biowhittaker) were added and incubations were made at 37 ° C with 5% CO2. The growth medium of the MAC-T line also contained 5 µg of insulin / ml and 1 µg of hydrocortisone / ml. S. aureus 2386, its mutants by deletion of katA, hlb and katA / hlb , as well as the three complemented mutants, were grown for 18-20 hours in BHI medium at 37 ° C with shaking. The cultures were centrifuged, washed twice with saline phosphate buffer (PBS) pH 7, resuspended in PBS + 20% Glycerol and stored at -80 ° C in 1 ml aliquots. The bacterial concentration of the aliquots was determined after freezing by sowing serial dilutions in BHI plates. To prepare the bacterial suspensions, the necessary aliquots were thawed, washed twice with PBS pH 7 and resuspended at the desired concentration (3-5 x 105 colony forming units / ml (cfu / ml)) in invasion medium (medium cell culture without antibiotics). Intracellular survival assays were performed in 24-well plates. The wells were inoculated with 3 x 10 4 cells and incubated for 48 hours under the conditions described above. Approximately 16 hours before the test, the cells were washed with sterile PBS and the culture medium was replaced by invasion. Before the experiment, the cells present in one of the wells were counted to check that there were 5-6 x 10 4 cells / well. For the experiment, new invasion medium was added to the cells and the wells were inoculated with the bacterial suspensions at a multiplicity of infection of 10: 1 (J774A.1) or 50: 1 (MAC-T). The plates were then centrifuged at 1500 rpm for 5 minutes and incubated for 1 hour at 37 ° C with 5% CO2. After this incubation, the medium was removed, the cells were washed twice with sterile PBS and invasion medium with 100 µg of gentamicin / ml was added to each well to destroy the extracellular bacteria. After incubation with gentamicin for 1 (internalized bacteria), 4, 8 and 24 hours, two washes were performed with sterile PBS and the infected cells were lysed by adding 0.2% Triton x-100. Serial dilutions of the lysates were made and seeded in BHI plates to make the cfu count. The surviving intracellular bacteria at 4, 8 and 24 hours were expressed as a percentage of the intracellular bacteria present in the first hour.
El comportamiento tanto de la cepa parental de S. aureus, como de los mutantes por deleción de katA y de hlb fue análogo hallándose una disminución progresiva de las bacterias intracelulares viables. En el análisis de varianza no se encontraron diferencias significativas tras 4, 8 y 24 horas postinfección.The behavior of both the parental strain of S. aureus , and of the mutants by deletion of katA and hlb was similar, finding a progressive decrease in viable intracellular bacteria. In the analysis of variance no significant differences were found after 4, 8 and 24 hours post-infection.
Sin embargo, al comparar los resultados obtenidos en los estudios de supervivencia intracelular de S. aureus 2386 y su doble mutante katA/hlb (S. aureus CECT 7061), se constató que, contrariamente al comportamiento mostrado por los mutantes simples, el doble mutante presentaba mayor proliferación a las 4 horas postinfección en MAC-T y una mayor persistencia intracelular que la cepa parental: en J774A.1 a las 4, 8 y 24 horas postinfección, y en MAC-T a las 8 y 24 horas. Además, se encontró que estas diferencias eran estadísticamente significativas en el análisis de varianza en los tres tiempos considerados. La prueba de Tukey-Kramer reveló la existencia de diferencias significativas entre la cepa parental y el doble mutante, lo que implica que las diferencias observadas se deben a la deleción de estos dos genes.However, when comparing the results obtained in the intracellular survival studies of S. aureus 2386 and its double katA / hlb mutant ( S. aureus CECT 7061), it was found that, contrary to the behavior shown by the single mutants, the double mutant it presented greater proliferation at 4 hours post-infection in MAC-T and greater intracellular persistence than the parental strain: in J774A.1 at 4, 8 and 24 hours post-infection, and in MAC-T at 8 and 24 hours. In addition, it was found that these differences were statistically significant in the analysis of variance in the three times considered. The Tukey-Kramer test revealed the existence of significant differences between the parental strain and the double mutant, which implies that the differences observed are due to the deletion of these two genes.
Cálculo de las dosis letales 50 (DL_{50}) en ratón. Con el fin de evaluar el efecto en la virulencia de los tres mutantes, se estimó la dosis letal cincuenta (DL_{50}) tras inocular ratones por vía intraperitoneal, tal y como se detalla a continuación.Calculation of lethal doses 50 (DL 50) in mouse. In order to evaluate the effect on the virulence of the three mutants, the lethal dose fifty (DL 50) was estimated after inoculate mice intraperitoneally, as detailed in continuation.
S. aureus 2386 y sus mutantes por deleción de katA, hlb y katA/hlb se cultivaron durante 18-20 horas en 100 ml de medio BHI a 37ºC con agitación. Los cultivos se centrifugaron, se lavaron dos veces con PBS estéril y se resuspendieron en 5 ml de PBS estéril para obtener la dosis de inoculación más alta. Las demás dosis se obtuvieron mediante diluciones seriadas de ésta. Las dosis utilizadas oscilaron entre 1x10^{6} y 1x10^{11} ufc/ml. Las ufc se confirmaron mediante la siembra de diluciones seriadas en placas de BHI. Para el ensayo se utilizaron ratones Swiss hembras de 4 semanas de edad (21-23 g), que se dispusieron en 5 grupos de 5 animales cada uno y a los que se suministró agua y comida ad libitum bajo condiciones normales de luz y temperatura. Los animales se inocularon intraperitonealmente con 0,4 ml de las suspensiones bacterianas y se registraron las muertes producidas cada 24 horas durante 7-10 días. Las DL_{50} se calcularon por el método de Reed y Muench (1938. Am J Hyg. 27: 493-497). S. aureus 2386 and its mutants by deletion of katA, hlb and katA / hlb were grown for 18-20 hours in 100 ml of BHI medium at 37 ° C with stirring. The cultures were centrifuged, washed twice with sterile PBS and resuspended in 5 ml of sterile PBS to obtain the highest inoculation dose. The other doses were obtained by serial dilutions. The doses used ranged from 1x10 6 to 1x10 11 cfu / ml. The cfu were confirmed by sowing serial dilutions in BHI plates. For the test, 4-week-old Swiss female mice (21-23 g) were used, which were arranged in 5 groups of 5 animals each and to which water and food were supplied ad libitum under normal conditions of light and temperature. Animals were inoculated intraperitoneally with 0.4 ml of bacterial suspensions and deaths occurred every 24 hours for 7-10 days. The DL_ {50} were calculated by the method of Reed and Muench (1938. Am J Hyg. 27: 493-497).
La DL_{50} del mutante por deleción de katA fue inferior a la de la cepa silvestre S. aureus 2386, si bien en el análisis de varianza estas diferencias no fueron estadísticamente significativas. Por lo tanto, la catalasa por sí sola no es un factor que intervenga significativamente en la virulencia.The LD50 of the mutant by deletion of katA was lower than that of the wild strain S. aureus 2386, although in the analysis of variance these differences were not statistically significant. Therefore, catalase alone is not a factor that significantly intervenes in virulence.
Tampoco se encontraron diferencias significativas entre las DL_{50} del mutante por deleción del gen hlb y la cepa parental. Lo que significa que la \beta-toxina, por sí misma, tampoco tiene un papel significativo en la patogenicidad de S. aureus.Nor were significant differences found between the LD50 of the mutant by deletion of the hlb gene and the parental strain. Which means that β-toxin, by itself, also has no significant role in the pathogenicity of S. aureus .
Sin embargo, la atenuación del doble mutante katA/hlb sí fue significativa dado que se observó un incremento de 5 veces en la DL_{50} del mutante con respecto a la cepa parental (véase figura 3).However, the attenuation of the double mutant katA / hlb was significant since a 5-fold increase in the LD50 of the mutant with respect to the parental strain was observed (see Figure 3).
<110> Universidad Complutense de Madrid<110> Complutense University of Madrid
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\ hskip-.1em \ dddseqskipagcactattt caccatcatt atcactcctt
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\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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<210> 3<210> 3
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 30<211> 30
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> primer<213> first
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 3<400> 3
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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<210> 4<210> 4
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<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> primer<213> first
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 4<400> 4
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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<213> primer<213> first
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<400> 5<400> 5
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<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> primer<213> first
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 6<400> 6
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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<210> 7<210> 7
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<211> 30<211> 30
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<212> DNA<212> DNA
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<400> 7<400> 7
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
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\ hskip-.1em \ dddseqskipgactatgtca taaatttgat atgtagtttc
\ hfill30
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<210> 8<210> 8
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<211> 21<211> 21
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<212> DNA<212> DNA
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<213> primer<213> first
\vskip1.000000\baselineskip\ vskip1.000000 \ baselineskip
\vskip0.400000\baselineskip\ vskip0.400000 \ baselineskip
<400> 8<400> 8
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\hskip-.1em\dddseqskipttccgctttg aaattttaaa t
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Claims (12)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200500809A ES2307351B2 (en) | 2005-04-07 | 2005-04-07 | STAFFYLOCOCCUS AUREUS LIVED LIVED CEPA AS A VACCINE IN RUMINANT MASTITIS. |
| PCT/ES2006/000164 WO2006106166A1 (en) | 2005-04-07 | 2006-04-06 | Attenuated live strain of staphylococcus aureus as a vaccine against bovine mastitis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200500809A ES2307351B2 (en) | 2005-04-07 | 2005-04-07 | STAFFYLOCOCCUS AUREUS LIVED LIVED CEPA AS A VACCINE IN RUMINANT MASTITIS. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ES2307351A1 ES2307351A1 (en) | 2008-11-16 |
| ES2307351B2 true ES2307351B2 (en) | 2009-06-17 |
Family
ID=37073109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ES200500809A Expired - Fee Related ES2307351B2 (en) | 2005-04-07 | 2005-04-07 | STAFFYLOCOCCUS AUREUS LIVED LIVED CEPA AS A VACCINE IN RUMINANT MASTITIS. |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2307351B2 (en) |
| WO (1) | WO2006106166A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011027956A2 (en) * | 2009-09-04 | 2011-03-10 | 주식회사이언메딕스 | Extracellular vesicles derived from gram-positive bacteria, and disease model using same |
-
2005
- 2005-04-07 ES ES200500809A patent/ES2307351B2/en not_active Expired - Fee Related
-
2006
- 2006-04-06 WO PCT/ES2006/000164 patent/WO2006106166A1/en not_active Ceased
Non-Patent Citations (4)
| Title |
|---|
| BRAMLEY, A. J., PATEL, A. H., O'REILLY, M. et al. Roles of alpha- toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland. Infection and Immunity. Agosto 1989, Vol. 57, N$^{o}$ 8, páginas 2489-2494. ISSN 0019-9567. * |
| COLEMAN, D., KNIGHTS, J., RUSSELL, R. et al. Insertional inactivation of the Staphylococcus aureus beta-toxin by bacteriophage phi-13 occurs by site- and orientation-specific integration of the phi-13 genome. Molecular Microbiology. 1991, Vol. 5, N$^{o}$ 4, páginas 933-939. ISSN 0950-382X. * |
| ODIERNO, L., RISATTI, G., CALZOLARI, A. et al. Pathogenicity in mice of Staphylococcus aureus mutants deficient in exoprotein synthesis. Veterinary Microbiology. Agosto 1994, Vol. 41, N$^{o}$ 3 páginas 249-258. ISSN 0378-1135. * |
| SANZ, R., MARÍN, I., RUIZ-SANTA-QUITERIA, J. A. et al. Catalase deficiency in Staphylococcus aureus subsp. anaerobius is associated with natural loss-of-function mutations within the structural gene. Microbiology. Febrero 2000, Vol. 146, N$^{o}$ 2, páginas 465-475. ISSN 1350-0872. * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2006106166A1 (en) | 2006-10-12 |
| ES2307351A1 (en) | 2008-11-16 |
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