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Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

Peton V, Bouchard DS, Almeida S, Rault L, Falentin H, Jardin J, Jan G, Hernandez D, François P, Schrenzel J, Azevedo V, Miyoshi A, Berkova N, Even S, Le Loir Y - Vet. Res. (2014)

Bottom Line: The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide.In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells.N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1253 STLO, 65 rue de Saint Brieuc, 35042, Rennes Cedex, France. vincent.peton@rennes.inra.fr.

ABSTRACT
S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization ofS. aureusN305 and comparison with RF122. A. Coagulase activity of RF122 and N305. A 108 CFU aliquot was prepared from an overnight culture of each strain, washed and re-suspended in rabbit (cu), bovine (bo) or goat (ca) plasma. After a 4-h incubation at 37 °C, the level of coagulation of observed by tilting the tubes. B. Cytotoxic effects of S. aureus RF122 and N305 supernatants on MAC-T cells. S. aureus strains were grown for 24 h in RPMI with 50 mM deferoxamine. Supernatants (or PBS for the control condition) were mixed in DMEM at a ratio of 1:1 and incubated with MAC-T cells. Cytotoxicity was assessed using the MTT test and relative viability was expressed with regard to PBS-treated cells. Each experiment was performed in triplicate, and differences between the groups were compared using Student’s t test. **P < 0.005.
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Fig3: Phenotypic characterization ofS. aureusN305 and comparison with RF122. A. Coagulase activity of RF122 and N305. A 108 CFU aliquot was prepared from an overnight culture of each strain, washed and re-suspended in rabbit (cu), bovine (bo) or goat (ca) plasma. After a 4-h incubation at 37 °C, the level of coagulation of observed by tilting the tubes. B. Cytotoxic effects of S. aureus RF122 and N305 supernatants on MAC-T cells. S. aureus strains were grown for 24 h in RPMI with 50 mM deferoxamine. Supernatants (or PBS for the control condition) were mixed in DMEM at a ratio of 1:1 and incubated with MAC-T cells. Cytotoxicity was assessed using the MTT test and relative viability was expressed with regard to PBS-treated cells. Each experiment was performed in triplicate, and differences between the groups were compared using Student’s t test. **P < 0.005.

Mentions: Based on the differences in gene content revealed by sequence analysis, phenotypic differences could be expected in terms of biofilm formation, coagulase activity, cytotoxicity and invasive capacities. These four properties were tested in vitro at the phenotype level (see Materials and methods for details). In line with the absence of differences in terms of the content of genes involved in biofilm formation, the two strains did not display any significant differences in phenotype. Indeed, RF122 and N305 revealed the same ability to form a biofilm on an abiotic surface, as determined by Crystal violet staining on a plastic surface (data not shown). By contrast, with respect to coagulase activity, the presence of vwbSbo5 (Newbould 305_0962) in the N305 genome (Figure 1C), whose product was also found in the N305 surface exposed protein (Additional file 2), was clearly associated with an increased range of coagulase activity. N305 indeed clotted rabbit, goat and bovine plasma, whereas RF122 only clotted rabbit plasma (Figure 3A). Furthermore, N305 displayed greater cytotoxicity on MAC-T cells than RF122 after co-incubation with their respective supernatants. The viability of cells incubated with the N305 supernatant was 89% lower than under control conditions, whereas the viability of cells incubated with the RF122 supernatant was 36% lower (Figure 3B). N305 supernatant also presented a higher ability to hydrolyse caseins as shown by the translucent halo around the well of N305 deposit (Additional file 3).Figure 3


Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

Peton V, Bouchard DS, Almeida S, Rault L, Falentin H, Jardin J, Jan G, Hernandez D, François P, Schrenzel J, Azevedo V, Miyoshi A, Berkova N, Even S, Le Loir Y - Vet. Res. (2014)

Phenotypic characterization ofS. aureusN305 and comparison with RF122. A. Coagulase activity of RF122 and N305. A 108 CFU aliquot was prepared from an overnight culture of each strain, washed and re-suspended in rabbit (cu), bovine (bo) or goat (ca) plasma. After a 4-h incubation at 37 °C, the level of coagulation of observed by tilting the tubes. B. Cytotoxic effects of S. aureus RF122 and N305 supernatants on MAC-T cells. S. aureus strains were grown for 24 h in RPMI with 50 mM deferoxamine. Supernatants (or PBS for the control condition) were mixed in DMEM at a ratio of 1:1 and incubated with MAC-T cells. Cytotoxicity was assessed using the MTT test and relative viability was expressed with regard to PBS-treated cells. Each experiment was performed in triplicate, and differences between the groups were compared using Student’s t test. **P < 0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230361&req=5

Fig3: Phenotypic characterization ofS. aureusN305 and comparison with RF122. A. Coagulase activity of RF122 and N305. A 108 CFU aliquot was prepared from an overnight culture of each strain, washed and re-suspended in rabbit (cu), bovine (bo) or goat (ca) plasma. After a 4-h incubation at 37 °C, the level of coagulation of observed by tilting the tubes. B. Cytotoxic effects of S. aureus RF122 and N305 supernatants on MAC-T cells. S. aureus strains were grown for 24 h in RPMI with 50 mM deferoxamine. Supernatants (or PBS for the control condition) were mixed in DMEM at a ratio of 1:1 and incubated with MAC-T cells. Cytotoxicity was assessed using the MTT test and relative viability was expressed with regard to PBS-treated cells. Each experiment was performed in triplicate, and differences between the groups were compared using Student’s t test. **P < 0.005.
Mentions: Based on the differences in gene content revealed by sequence analysis, phenotypic differences could be expected in terms of biofilm formation, coagulase activity, cytotoxicity and invasive capacities. These four properties were tested in vitro at the phenotype level (see Materials and methods for details). In line with the absence of differences in terms of the content of genes involved in biofilm formation, the two strains did not display any significant differences in phenotype. Indeed, RF122 and N305 revealed the same ability to form a biofilm on an abiotic surface, as determined by Crystal violet staining on a plastic surface (data not shown). By contrast, with respect to coagulase activity, the presence of vwbSbo5 (Newbould 305_0962) in the N305 genome (Figure 1C), whose product was also found in the N305 surface exposed protein (Additional file 2), was clearly associated with an increased range of coagulase activity. N305 indeed clotted rabbit, goat and bovine plasma, whereas RF122 only clotted rabbit plasma (Figure 3A). Furthermore, N305 displayed greater cytotoxicity on MAC-T cells than RF122 after co-incubation with their respective supernatants. The viability of cells incubated with the N305 supernatant was 89% lower than under control conditions, whereas the viability of cells incubated with the RF122 supernatant was 36% lower (Figure 3B). N305 supernatant also presented a higher ability to hydrolyse caseins as shown by the translucent halo around the well of N305 deposit (Additional file 3).Figure 3

Bottom Line: The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide.In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells.N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1253 STLO, 65 rue de Saint Brieuc, 35042, Rennes Cedex, France. vincent.peton@rennes.inra.fr.

ABSTRACT
S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

No MeSH data available.


Related in: MedlinePlus