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Treatment of Staphylococcus aureus infections has become increasingly challenging due to the rapid emergence and dissemination of methicillin-resistant strains. In addition, S. aureus reside within biofilms at the site of infection. Few novel antibacterial agents have been developed in recent years and their bacteriostatic or bactericidal activity results in selective pressure, inevitably inducing antimicrobial resistance. Consequently, innovative antimicrobials with other modes of action are urgently needed. One alternative approach is targeting the bacterial quorum sensing (QS) system. Hamamelitannin (2′,5-di-O-galloyl-d-hamamelose; HAM) was previously suggested to block QS through the TraP QS system and was shown to increase S. aureus biofilm susceptibility towards vancomycin (VAN) although mechanistic insights are still lacking. In the present study we provide evidence that HAM specifically affects S. aureus biofilm susceptibility through the TraP receptor by affecting cell wall synthesis and extracellular DNA release of S. aureus. We further provide evidence that HAM can increase the susceptibility of S. aureus biofilms towards different classes of antibiotics in vitro. Finally, we show that HAM increases the susceptibility of S. aureus to antibiotic treatment in in vivo Caenorhabditis elegans and mouse mammary gland infection models.

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.

BackgroundMurine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.MethodsIntraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.ResultsMice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.ConclusionsThe current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0860-x) contains supplementary material, which is available to authorized users.