Staphylococcus aureus is a major pathogen that is responsible for mastitis in dairy herds. S. aureus mastitis is difficult to treat and prone to recurrence despite antibiotic treatment. The ability of S. aureus to invade bovine mammary epithelial cells (bMEC) is evoked to explain this chronicity. One sustainable alternative to treat or prevent mastitis is the use of lactic acid bacteria (LAB) as mammary probiotics. In this study, we tested the ability of Lactobacillus casei strains to prevent invasion of bMEC by two S. aureus bovine strains, RF122 and Newbould305, which reproducibly induce acute and moderate mastitis, respectively. L. casei strains affected adhesion and/or internalization of S. aureus in a strain-dependent manner. Interestingly, L. casei CIRM-BIA 667 reduced S. aureus Newbould305 and RF122 internalization by 60 to 80%, and this inhibition was confirmed for two other L. casei strains, including one isolated from bovine teat canal. The protective effect occurred without affecting bMEC morphology and viability. Once internalized, the fate of S. aureus was not affected by L. casei . It should be noted that L. casei was internalized at a low rate but survived in bMEC cells with a better efficiency than that of S. aureus RF122. Inhibition of S. aureus adhesion was maintained with heat-killed L. casei , whereas contact between live L. casei and S. aureus or bMEC was required to prevent S. aureus internalization. This first study of the antagonism of LAB toward S. aureus in a mammary context opens avenues for the development of novel control strategies against this major pathogen.

Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.

Staphylococcus aureus is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. S. aureus naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that S. aureus Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs in vitro with a designated protein content. Purified S. aureus N305-secreted EVs were not cytotoxic when tested in vitro on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live S. aureus N305. The in vivo immune response to purified S. aureus N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live S. aureus N305, heat-killed S. aureus N305 and to S. aureus lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live S. aureus induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, S. aureus N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary S. aureus N305-secreted EVs was comparable to that of heat-killed S. aureus N305 and, partly, by LTA. These results demonstrated that S. aureus N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined in vitro and in vivo data suggest that EVs are worth to be investigated to better understand the S. aureus pathogenesis and are relevant tools to develop strategies against bovine S. aureus mastitis.

S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection. We employed genomic, transcriptomic and proteomic approaches to comprehensively compare two clonally related S. aureus strains that reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical mastitis cases. Our data are consistent with a dose-dependant role of some staphylococcal factors in the hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins and iron acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity.

Background: Aspergillus fumigatus, a saprophytic mould, is responsible for life-threatening, invasive pulmonary diseases in immunocompromised hosts. The role of the airway epithelium involves a complex interaction with the inhaled pathogen. Antimicrobial peptides with direct antifungal and chemotactic activities may boost antifungal immune response.Results: The inducible expression of defensins by human bronchial epithelial 16HBE cells and A549 pneumocyte cells exposed toA. fumigatuswas investigated. Using RT-PCR and real time PCR, we showed an activation of hBD2 and hBD9 defensin genes: the expression was higher in cells exposed to swollen conidia (SC), compared to resting conidia (RC) or hyphal fragments (HF). The kinetics of defensin expression was different for each one, evoking a putative distinct function for each investigated defensin. The decrease of defensin expression in the presence of heat-inactivated serum indicated a possible link between defensins and the proteins of the host complement system. The presence of defensin peptide hBD2 was revealed using immunofluorescence that showed a punctual cytoplasmic and perinuclear staining. Quantification of the cells stained with anti hBD2 antibody demonstrated that SC induced a greater number of cells that synthesized hBD2, compared to RC or HF. Labelling of the cells with anti-hBD-2 antibody showed a positive immunofluorescence signal around RC or SC in contrast to HF. This suggests co-localisation of hBD2 and digested conidia. The HBD2 level was highest in the supernatants of cells exposed to SC, as was determined by sandwich ELISA. Experiments using neutralising anti-interleukine-1β antibody reflect the autocrine mechanism of defensin expression induced by SC. Investigation of defensin expression at transcriptional and post-transcriptional levels demonstrated the requirement of transcription as well as new protein synthesis duringA. fumigatusdefensin induction. Finally, induced defensin expression in primary culture of human respiratory cells exposed toA. fumigatuspoints to the biological significance of described phenomena.Conclusion: Our findings provide evidence that respiratory epithelium might play an important role in the immune response duringAspergillusinfection. Understanding the mechanisms of regulation of defensin expression may thus lead to new approaches that could enhance expression of antimicrobial peptides for potential therapeutic use during aspergillosis treatment.