WO2006106166A1 - Attenuated live strain of staphylococcus aureus as a vaccine against bovine mastitis - Google Patents

Abstract

The invention relates to the use of an attenuated live strain of Staphylococcus aureus as a vaccine against ruminant mastitis. More specifically, the invention relates to a novel live vaccine strain of S. aureus which, in addition to being attenuated, is characterized by the following more significant innovations, namely: greater proliferation rates and persistence in mammary epithelial cells. The aforementioned properties promote a prolonged immune response. Said strain has been deposited and registered in the Spanish Type Culture Collection (CECT) at the University of Valencia, Campus de Burjassot, Edificio de Investigación, 46100 Burjassot (Valencia), with reference number CECT 7061. The strategy used in the construction thereof confers complete genetic stability on the mutation. In addition, the vaccine strain lacks antibiotic resistance markers in order to fulfil the associated biological safety requirement.

Description

TITLE

Live attenuated strain of Staphylococcus aureus as a vaccine in ruminant mastitis.

TECHNICAL FIELD

The present invention falls within the field of Animal Health. More specifically, the invention refers to the production of a live strain of

Staphylococcus aureus attenuated by means of the deletion of two genetically stable genes, without antibiotic resistance markers and with the ability to proliferate and persist in mammary epithelial cells. It constitutes a new vaccine strain for ruminant mastitis.

STATE OF THE TECHNIQUE. BACKGROUND AND PROBLEM TO BE SOLVED

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 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.

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, in which it 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, also 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 the 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, walls isolated cell phones, 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 lnt J Cancer.; 75 (6): 873-7 .; Amorena, B., et al. 1994. Vaccine. 12 (3): 243-9 .; Nordhaug et al. ., 1994, J. Dairy ScL 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 it more expensive and difficult to produce ( Dietrich, G. et al, 2003. Curr Opin Mol Ther. 5 (1): 10-9. Review).

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 intramammary pathogenicity of S. aureus: surface components of the bacterium (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 Ia and β).

The β-toxin or β-hemolysin is a sphingomyelinase C dependent on Mg ²⁺ that degrades the sphingomyelin present in the outer layer of phospholipids of the cell membrane. The cell lysis 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 strains of S. aureus 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 participation of β-toxin in the pathogenesis of S. aureus, and specifically in breast infection, has not been conclusively demonstrated to date. Thus, in studies with purified toxin, it has been described that β-toxin induces 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 which is cytotoxic for bovine mammary epithelial cells in culture (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 were found in virulence 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 CeII Biol. 21 : 375-382).

S. aureus produces numerous extracellular enzymes such as hyaluronidase, nuclease, lipase, catalase, phosphatase, staphylokinase and proteases that have also been related to the pathogenicity of the bacterium 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.

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 it has not been precisely determined to date the possible role that catalase can play in its pathogenesis, if it plays. 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. Infecí 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 the bacteria in the liver of inoculated roots through an intravenous route and concluded that the absence of caylase activity did not decrease the virulence of S. aureus. The gene that encodes caialase (katA) in S. aureus has been sequenced and characterized (Sanz eí al. 2000. Microbiology. 146: 465-475).

Although S. aureus has been considered as an extracellular pathogen, several studies in vivo and in vitro have shown that it is capable of adhering and being internalized by a wide variety of cell ips, being able to survive and, sometimes, multiply in the interior of epicelial cells (Almeida e al. 1996. J. Dairy Sci. 79: 1021-1026; Bayles et al. 1998. Infect Immun. 66: 336-342; Kahl et al. 2000. Infeci Immun. 68: 5385-5392; Brouilleíí eí al. 2003. Microb Paíhog. 35: 159-168; Hess eí al. 2003. J Surg Res. 114: 42-49), endoíeliales (Hamill eí al. 1986. I infected Immun. 54: 833-836; Yao ei al. 1995. I infected Immun. 63: 1835-1839; Menzies and Kourteva. 1998. I infected Immun. 66: 5994-5998), fibroblasts (Fowler et al. 2000. Eur J CeII Biol. 79: 672-679), osseoblasios (Hudson et al. 1995. Microb Pathog. 19: 409-419; Ahmed eí al. 2001. I infected Immun. 69: 2872-2877) and even phagocytic cells (Gresham eí al. 2000. J Immunol. 164: 3713 -3722; Hébert eí al. 2000. FEMS Microbiol Leít. 193: 57-62; Brouillette et al. 2003. Microb Pathog. 35: 159-168). The internalization of S. aureus by the host cells can contribute to the persistence of the infection by providing a resistance resists the immune system and restrains the antibiotic irradiations (Ferens and Bohach. 2000. J Lab Clin Med. 135: 225-230 ). DESCRIPTION OF THE INVENTION

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.

Although the published information did not allow to venture the use of the katA and hlb genes as targets to build a live attenuated S. aureus strain 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.

The method used to carry out this invention meets the essential requirements of biological safety for new generation vaccines. On the one hand, it ensures the stability of the mutation since the reversion to the wild phenotype is totally impeded due to the absence of targets required for homologous or illegitimate recombination from natural populations of bacteria. On the other hand, the constructed strain lacks antibiotic resistance markers.

Despite the results known so far, surprisingly, the novel combination of the two deletions of the katA and hlb genes provides this strain with 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.

The double mutation has a clear effect increasing the proliferation and persistence of the mutant in the MAC-T cell line of mammary epithelial cells and the persistence in the cell line of macrophages J774A.1, observing a synergistic and potentiating effect between both mutations (see Figures 4 and 5). The proliferation of the muíaeia is more lenía than the one of the parental strain, reaching the maximum of viable níracellular bacterae more far. The greater permissiveness for the replication of muierie in mammary epielial cells is a consequence of its lower toxicity for this cell type. 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, make double mutant a live attenuated vaccine against infections caused by S. aureus and more specifically against mastitis produced by this microorganism.

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.

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.

DESCRIPTION OF THE DRAWINGS

To facilitate the understanding of the main features of the invention and forming an integral part of this specification, a series of figures are attached. For illustrative and non-limiting purposes, the following has been represented:

Figure 1. Shows the scheme of the construction 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 Datábase). 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.

Figure 2. Scheme of the construction of the thermosensitive plasmid pERkat. The numbers 5, 6, 7 and 8 correspond to the nomenclature of the oligonucleotides described in SEQ ID NO: 5, 6, 7 and 8, respectively. Figure 3. DL ₅₀ trials in the murine model. The arithmetic averages of the number of cfu necessary to produce the 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) for S. aureus 2386 (2.80E + 9) ± DS 2.33E + 9; (2) for its double mutant katA / hlb, S. aureus CECT 7061, (1.41 E + 10) ± DS 4.68E + 9; and (3) for said mutant supplemented with the plasmid containing both 5.77E + 8 ± DS 3.11 E + 8 genes.

Figure 4. Intracellular survival of S. aureus 2386, its double katA / hlb mutant (S. aureus CECT 7061) and said mutanie supplemented with the plasmid containing both genes, in the macrophage cell line J774A.1 for 24 hours. With a rhombus, strain 2386 is represented, with a square the double mutant and a double triangle mediated the complementary double muanie. In the ordinates axis, the percentage (%) of viable intracellular bacteria was repressed. This damage corresponds to the mean number of viable bacteria, plus-minus its standard deviation (± SD), expressed in percentage referred to the total of bacteria present in the intracellular environment at time zero. The time (T) in hours (h) is indicated on the abscissa axis.

Figure 5. Intracellular survival of S. aureus 2386, its double muierie katA / hlb (S. aureus CECT 7061) and said muierie supplemented with the plasmid containing both genes, in the MAC-T mammary epithelial cell line. In the axes of abscissa and ordinate, the same variables were represented as in Figure 4. Likewise, the different strains are indicated by the same symbols as in the previous figure.

EMBODIMENT OF THE INVENTION

Strains of S. aureus and E. coli used routinely grown at 37 ⁰ C with shaking in BHI medium (Brain Heart Infusion) and LB (Luria-Bertani), respecíivameníe. Strains transformed with plasmids thermosensitive incubated at 32 ⁰ C for eviíar the loss of the plasmid. The media were supplemented with erythromycin (5μg / ml) when necessary. To obtain solid medium agar (15g / l) was added.

S. aureus 2386 was used as a wild strain to consuire the Muiah strains On the other hand, in the tests carried out 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.

Having described the present invention, it is further illustrated by the following examples, which are not limiting of its scope, which is defined exclusively by the attached claim note.

Example 1: 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 per oral, 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 in 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 Gótz et al. (1981. J Bacteriol. 145: 74-81). Transformant bacteria, verified by PCR, were incubated at 32 ⁰ C in the presence of erythromycin until the stationary growth phase. Then the temperature was raised to 43 ⁰ C for 6 hours to induce the homologous recombination between the plasmid and the bacterial chromosome. Subsequently subcultures were performed at 37 ⁰ 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, the deletion of the hlb gene was confirmed by PCR techniques.

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 they were contacted with 3% hydrogen peroxide. Likewise, the deletion of the katA gene was checked by PCR.

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 esíraiegia described for the mutant by deletion of katA. This double muierie is deposited and registered in the Spanish Type Culinary Collection (CECT), University of Valencia, Burjassoí Campus, Research Building, 46100 Burjassoí (Valencia), with reference CECT 7061.

Finally, all the mutani were supplemented using the plasmid pHpS9, in which the genes hlb (pHhlb), katA (pHkaí) and both (pHkaíhlb) were cloned. In all the cases, the complemeníación reproduced in the muiáíes the phenoíipo of the silvesíre strain from which they derived, reason why the observed phenotype was associated to the specific deletion of the gene.

Example 2: Intracellular survival assays in J774A.1 and MAC-T cell lines. To study whether the loss of caylase activity, the lack of bioxine production or the absence of the two acivities affected survival and in vitro proliferation of S. aureus, intracellular survival studies were conducted in two different cell lines: macrophages J774A.1 and MAC-T mammary epithelial cells. The macrophages constitute one of the essential pieces of innate immunity, and participate actively in the presence of anigens to induce a satisfactory acquired immunity. Mammary epiyelia cells were used as an in vitro study model of the behavior of S. aureus in the mammary gland of ruminants.

The J774A.1 and MAC-T cell lines were handled with Dulbecco's modified Eagle medium (DMEM) to which 10% fetal bovine serum (FBS) and 1% Pen / Sírep / Fungizones Mix (Biowhiíker) were added and incubations were done at 37 ⁰ 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 muiáíes by deletion of katA, hlb and katA / hlb, as well as the complemeníados mutaníes íres, were cultivated during 18-20 hours in medium BHI to 37 ⁰ C with agiíación. The culíivos were centrifuged, washed twice with iampon phosphaio saline (PBS) pH 7, were resuspended in PBS + 20% Glycerol and stored at -8O ⁰ 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 verify that there were 5-6 x 10 ⁴ cells / well. For the experiment, new invasion media 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). Then the plates were centrifuged at 1500 rpm for 5 minutes and incubated for 1 hour at 37 ⁰ C with 5% CO _2. 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 used adding 0.2% Triton x-100. Serial dilutions of those used were made and seeded on BHI plates to make the ufe 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.

The behavior of both the parental strain of S. aureus, and 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.

However, when comparing the results obtained in the intracellular survival studies of S. aureus 2386 and its double mutant katA / hlb (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. Also I know He 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 observed differences are due to the deletion of these two genes.

Example 3: Calculation of lethal doses 50 (LD ₅₀ ) in mice. In order to evaluate the effect on virulence of the three mutants, the lethal dose fifty (LD ₅₀ ) was estimated after inoculating mice intraperitoneally, as detailed below.

S. aureus 2386 and its deletion mutants katA, and hlb katA / hlb cultured for 18-20 hours in 100 ml of BHI medium at 37 ⁰ 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 ufe 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. The 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).

The DL ₅₀ 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 esistically significant. On the other hand, the Calaslasa by itself is not a factor that significantly implies virulence.

Nor were significant differences found between the DL ₅₀ of the muaniae by deletion of the hlb gene and the parenchymal strain. Which means that β-ioxin, by itself, has a significant role in the pathogenicity of S. aureus.

However, the attenuation of the double mutation katA / hlb was significant given that a 5-fold increase in the LD50 of the mutant with respect to the parental strain was observed (see Figure 3).

Claims

1.- Live attenuated strain of Staphylococcus aureus characterized in that it lacks catalase activity and beta toxin. 2. Live attenuated strain of Staphylococcus aureus, according to claim 1, characterized by the deletion of the katA and hlb genes encoding the enzymes responsible for catalase activity and beta toxin, respectively. 3. Live attenuated strain of Staphylococcus aureus, according to claim 2, characterized in that the deletion of the katA and hlb genes is a phase deletion. 4.- Live attenuated strain of Staphylococcus aureus, according to claim 3, characterized in that the deletion in the katA and hlb phase is carried out by means of a strategy that avoids the reversal of the mutated strain to the wild phenotype. 5.- Live attenuated strain of Staphylococcus aureus, according to claim 4, characterized in that the strategy chosen to prevent the reversal of the mutated strain to the wild phenotype is the double homologous recombination. 6. Live attenuated strain of Staphylococcus aureus, according to previous claims, characterized in that the deletion of katA and hlb does not leave in the bacterium selection markers for antibiotic resistance. 7.- Live attenuated strain of Staphylococcus aureus, according to previous claims, characterized in that it has a greater capacity for proliferation and persistence in epithelial cells of the mammary gland than the parental strain. 8.- Live attenuated strain of Staphylococcus aureus, according to previous claims, wherein the strain is Staphylococcus aureus CECT 7061. 9. Use of a live attenuated strain of Staphylococcus aureus, as described in the preceding claims, as a vaccine against infectious diseases caused by Staphylococcus aureus. 10. Use of an attenuated live strain of Staphylococcus aureus, as described in claims 1 to 8, as a vaccine against infectious diseases caused by Staphylococcus aureus in the mammary gland of ruminants. 11. A vaccination kit for mammals against infectious diseases caused by Staphylococcus aureus comprising a container that includes a live attenuated strain as described in claims 1 to 8. 12. Use of a live attenuated strain of Staphylococcus aureus, according to claims 1 to 8, as a vaccine vehicle for heterologous antigens.