Daptomycin is a last-resort membrane-targeting lipopeptide approved for the treatment of drug-resistant staphylococcal infections, such as bacteremia and implant-related infections. Although cases of resistance to this antibiotic are rare, increasing numbers of clinical,in vitro, and animal studies report treatment failure, notably againstStaphylococcus aureus. The aim of this study was to identify the features of daptomycin and its target bacteria that lead to daptomycin treatment failure. We show that daptomycin bactericidal activity againstS. aureusvaries significantly with the growth state and strain, according to the membrane fatty acid composition. Daptomycin efficacy as an antibiotic relies on its ability to oligomerize within membranes and form pores that subsequently lead to cell death. Our findings ascertain that daptomycin interacts with tolerant bacteria and reaches its membrane target, regardless of its bactericidal activity. However, the final step of pore formation does not occur in cells that are daptomycin tolerant, strongly suggesting that it is incapable of oligomerization. Importantly, membrane fatty acid contents correlated with poor daptomycin bactericidal activity, which could be manipulated by fatty acid addition. In conclusion, daptomycin failure to treatS. aureusis not due to a lack of antibiotic-target interaction, but is driven by its capacity to form pores, which depends on membrane composition. Manipulation of membrane fluidity to restoreS. aureusdaptomycin bactericidal activityin vivocould open the way to novel antibiotic treatment strategies.

Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections (BAI), and the choice of antibiotics to treat these infections remains a challenge for the medical community. In particular, daptomycin has been reported to fail against implant-associated S. aureus infections in clinical practice, while its association with rifampin remains a good candidate for BAI treatment. To improve our understanding of such resistance/tolerance toward daptomycin, we took advantage of the dynamic fluorescence imaging tools (time-lapse imaging and fluorescence recovery after photobleaching [FRAP]) to locally and accurately assess the antibiotic diffusion reaction in methicillin-susceptible and methicillin-resistant S. aureus biofilms. To provide a realistic representation of daptomycin action, we optimized an in vitro model built on the basis of our recently published in vivo mouse model of prosthetic vascular graft infections. We demonstrated that at therapeutic concentrations, daptomycin was inefficient in eradicating biofilms, while the matrix was not a shield to antibiotic diffusion and to its interaction with its bacterial target. In the presence of rifampin, daptomycin was still present in the vicinity of the bacterial cells, allowing prevention of the emergence of rifampin-resistant mutants. Conclusions derived from this study strongly suggest that S. aureus biofilm resistance/tolerance toward daptomycin may be more likely to be related to a physiological change involving structural modifications of the membrane, which is a strain-dependent process. Staphylococcus aureus is a Gram-positive bacterial species shown to be the most frequent cause of biofilm-associated infections (BAI) (1) and one of the major causes of morbidity and mortality in hospitals and communities (2). Unlike planktonic cells, biofilms exhibit specific phenotypic traits allowing them to resist host defenses and antibiotic treatments (3), which frequently leads to chronic infections such as endocarditis, sinusitis, and osteomyelitis and also to implant-associated infections (4).Among the most recent clinically used antibiotics, daptomycin is a cyclic lipopeptide approved for the treatment of serious staphylococcal infections such as bacteremia and implant-related infections (5). Daptomycin is a calcium-dependent antibiotic that acts by insertion into the Gram-positive cytoplasmic membranes where it forms oligomeric pores, causing potassium ion leakage and subsequent membrane depolarization, leading ultimately to cell death (6). As is the case for many antibiotics, daptomycin has been shown to exhibit a significant bactericidal activity against planktonic cells (7-9). However, the eradication of adherent bacteria is rarely achieved despite the large number of in vitro and animal studies in which daptomycin activity was evaluated (10-16). Besides the results of the literature that appear controversial (17), direct comparison between studies is not directly possible, since the biofilm growth and treatment protocols...

Despite the heterogeneity of results according to bacterial strains, these innovative models represent an option to better evaluate the in vitro efficacy of antibiotics on Dacron(®)-related biofilm S. aureus infections, and to screen different antibiotic regimens in a mouse model of PVGIs.

Carbon Dots (CDs) are innovative materials which have potential applications in many fields, including nanomedicine, energy and catalysis.

Fast and selective detection of pathogens represents high challenges given the considerably high proliferation rate and mutation potential of bacteria against antibiotics. With this aim, anionic red-orangeemitting fluorescent organic nanoparticles (FONs), characterized by high brightness and photostability, are developed to selectively stain Staphylococcus aureus after only 5 min of exposure. No cytotoxicity effects are observed as a result of the negatively charge surface of the employed FONs. By contrast, no staining can be observed with Pseudomonas aeruginosa strains. The exclusive labeling of Gram-positive bacteria is ascribed to originate from the phosphonic acid moieties incorporated in the FON-constituting fluorophores because model FONs, devoid of phosphonic acids, show no adhesion under the same experimental conditions. Tight hydrogen bonding between the FON acidic units and the peptidoglycan (PG) layers comprising the outer wall of S. aureus is suspected to be the prevailing factor for the encountered selective interactions. PG layers from S. aureus are employed to apprehend the interactions developed between FONs and the bacteria membrane. Correlative light electron microscopy using confocal fluorescence microscopy and SEM reveal FONs mainly located at extensively reorganized or "dented" PG areas. Such privileged localizations tend to suggest multivalent physicochemical interactions to aggregate a multifold of nanoparticles. Finally, spectral follow-up of the FON-stained bacteria membrane shows significant hypsochromic shift of the fluorescence emission, signaling progressive disassembly of FONs and a change of the surrounding polarity. This feature offers promising perspectives to use doped FONs as theranostic agents to liberate encapsulated antibiotics upon FON disintegration inside the bacteria membrane.

We took benefit from Atomic Force Microscopy (AFM) in the force spectroscopy mode to describe the time evolution -over 24h-of the surface nanotopography and mechanical properties of the strain Staphylococcus aureus 27217 from bacterial adhesion to the first stage of biofilm genesis. In addition, Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) experiments allowed identifying two types of self-adhering subpopulations (the so-called "bald" and "hairy" cells) and revealed changes in their relative populations with the bacterial culture age and the protocol of preparation. We indeed observed a dramatic evanescing of the "hairy" subpopulation for samples that underwent centrifugation and resuspension processes. When examined by AFM, the "hairy" cell surface resembled to a herringbone structure characterized by upper structural units with lateral dimensions of ~70 nm and a high Young modulus value (~ 2.3 MPa), a mean depth of the trough between them of ~15nm and a resulting roughness of ~5nm. By contrast, the "bald" cells appeared much softer (~ 0.35 MPa) with a roughness one order of magnitude lower. We observed too the gradual detachment of the herringbone patterns from the "hairy" bacterial envelope of cell harvested from a 16h old culture and their progressive accumulation between the bacteria in the form of globular clusters. The secretion of a soft extracellular polymeric substance was also identified that, in addition to the globular clusters, may contribute to the initiation of the biofilm spatial organization.

A blue luminescent and superhydrophobic coating based on an electropolymerized fluorinated-pyrene monomer and its planktonic bacteria and biofilm repellent properties are reported. Two different pathogenic bacterial strains (Gram-positive and Gram-negative) at two different incubation times (2 h planktonic bacterial and 24 h biofilm adhesion) were studied and monitored (analyzed) using multicolor scanning confocal fluorescence microscopy. The coating was proved to reduce bacterial adhesion by 65%. It is highly effective against biofilm attachment, with 90% reduction of bacteria surface coverage. This blue fluorescent surface provides a facile method to characterize the coating, observe the bacterial distribution and quantify the bacterial coverage rate by fluorescence imaging of different colors. Furthermore, the film does not show significant bacterial toxicity during the working incubation times.

Staphylococcus aureus is one of the most frequent pathogens responsible for biofilm-associated infections. Among current clinical antibiotics, very few enable long-term successful treatment. Thus, it becomes necessary to better understand antibiotic failures and successes in treating infections in order to master the use of proper antibiotic therapies. In this context, we took benefit from a set of fluorescence spectroscopy and imaging methods, with the support of conventional microbiological tools to better understand the vancomycin-rifampin combination (in)efficiency against S. aureus biofilms. It was shown that both antibiotics interacted by forming a complex. This latter allowed a faster penetration of the drugs before dissociating from each other to interact with their respective biological targets. However, sufficiently high concentrations of free vancomycin should be maintained, either by increasing the vancomycin concentration or by applying repetitive doses of the two drugs, in order to eradicate rifampin-resistant mutants.