Design of Triazolium-Grafted Peptidomimetic Macrocycles with Facial Amphipathicity to Target Pathogenic Bacteria.

Access to 1,2,3-triazolium-grafted peptoid macrocycles was developed by macrocyclization and multivalent postmodification of linear peptoid oligomers carrying an alternance of benzylic and propargyl groups as side chains. X-ray analysis and NMR studies revealed a conformational preference for constrained hairpin-shaped structures leading to the facial amphipathic character of these macrocycles. A preliminary evaluation showed the antimicrobial activities of these new cationic amphipathic architectures.

Elemental sulfur enhances the anti-fungal effect of Lacticaseibacillus rhamnosus Lcr35.

Lacticaseibacillus rhamnosus Lcr35 is a well-known bacterial strain whose efficiency in preventing recurrent vulvovaginal candidiasis has been largely demonstrated in clinical trials. The presence of sodium thiosulfate (STS) has been shown to enhance its ability to inhibit the growth of Candida albicans strains. In this study, we confirmed that Lcr35 has a fungicidal effect not only on the planktonic form of C. albicans but also on other life forms such as hypha and biofilm. Transcriptomic analysis showed that the presence of C. albicans induced a metabolic adaptation of Lcr35 potentially associated with a competitive advantage over yeast cells. However, STS alone had no impact on the global gene expression of Lcr35, which is not in favor of the involvement of an enzymatic transformation of STS. Comparative HPLC and gas chromatography-mass spectrometry analysis of the organic phase from cell-free supernatant (CFS) fractions obtained from Lcr35 cultures performed in the presence and absence of STS identified elemental sulfur (S0) in the samples initially containing STS. In addition, the anti-Candida activity of CFS from STS-containing cultures was shown to be pH-dependent and occurred at acidic pH lower than 5. We next investigated the antifungal activity of lactic acid and acetic acid, the two main organic acids produced by lactobacilli. The two molecules affected the viability of C. albicans but only at pH 3.5 and in a dose-dependent manner, an antifungal effect that was enhanced in samples containing STS in which the thiosulfate was decomposed into S0. In conclusion, the use of STS as an excipient in the manufacturing process of Lcr35 exerted a dual action since the production of organic acids by Lcr35 facilitates the decomposition of thiosulfate into S0, thereby enhancing the bacteria's own anti-fungal effect.

Role of Klebsiella pneumoniae Type VI secretion system (T6SS) in long-term gastrointestinal colonization.

Type VI secretion systems (T6SS), recently described in hypervirulent K. pneumoniae (hvKp) strains, are involved in bacterial warfare but their role in classical clinical strains (cKp) has been little investigated. In silico analysis indicated the presence of T6SS clusters (from zero to four), irrespective of the strains origin or virulence, with a high prevalence in the K. pneumoniae species (98%). In the strain CH1157, two T6SS-apparented pathogenicity islands were detected, T6SS-1 and -2, harboring a phospholipase-encoding gene (tle1) and a potential new effector-encoding gene named tke (Type VI Klebsiella effector). Tle1 expression in Escherichia coli periplasm affected cell membrane permeability. T6SS-1 isogenic mutants colonized the highest gastrointestinal tract of mice less efficiently than their parental strain, at long term. Comparative analysis of faecal 16S sequences indicated that T6SS-1 impaired the microbiota richness and its resilience capacity. Oscillospiraceae family members could be specific competitors for the long-term gut establishment of K. pneumoniae.

Lacticaseibacillus rhamnosus Lcr35 Stimulates Epithelial Vaginal Defenses upon Gardnerella vaginalis Infection.

Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria, such as Gardnerella vaginalis, and low levels of "healthy" lactobacilli leads to bacterial vaginosis (BV), usually associated with a low-grade inflammatory process. Despite appropriate antibiotic treatment, G. vaginalis-associated BV is characterized by significant recurrence. The use of probiotics could be an interesting alternative therapy due to their ability to rebalance vaginal microbiota. In this study, we investigated the effects of a well-characterized probiotic strain, Lacticaseibacillus rhamnosus Lcr35, on epithelial vaginal and dendritic cell (DC) immune responses after G. vaginalis infection. In an in vitro coculture model with human monocyte-derived dendritic cells and a vaginal epithelial cell (VEC) monolayer, the Lcr35 strain induced DC activation, as evidenced by the induction of maturation and synthesis of interleukin-8 (IL-8) and CCL-20 chemokines upon apical challenge of the VECs by G. vaginalis. Analysis of the vaginal epithelial response showed that the presence of Lcr35 significantly increased the production of the proinflammatory cytokines IL-8 and IL-1ß and human ß-defensin 2 (HBD-2), whereas the concentration of secretory leukocyte protease inhibitor (SLPI) was decreased in G. vaginalis-infected vaginal epithelial cells. Treatment with recombinant SLPI was associated with upregulation of Lcr35-stimulated IL-8 and HBD-2 production. These results suggest that inhibition of SLPI by Lcr35 in vaginal epithelial cells contributes to the host defense response against G. vaginalis infection.

Plasmidome analysis of a hospital effluent biofilm: Status of antibiotic resistance.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

Tick ecology and Lyme borreliosis prevention: a regional survey of pharmacists' knowledge in Auvergne-Rhône-Alpes, France.

The most prevalent vector-borne diseases in Europe are caused by tick-borne pathogens, such as bacteria of the genus Borrelia that cause Lyme borreliosis. In this context, retail pharmacists are frequently the first medical source of information in the event of a tick bite. The objective of this study was to assess pharmacy professionals' knowledge about both tick ecology and the appropriate measures for tick bites and Lyme borreliosis prevention. It was based on an online survey of 364 pharmacists and pharmacy assistants located in the Auvergne-Rhône-Alpes region of France. The results showed solid knowledge about preventive measures for tick bite and Lyme borreliosis, but weaker knowledge about tick biology (hosts, suitable habitats, favorable conditions for tick activity, etc.). In particular, several stereotypes were observed in the responses of the pharmacy professionals. These appear to result from a social construction of the knowledge on ticks and tick-borne diseases previously shown to the general population in the region. The results highlight the need for continuous training about ticks and tick-borne diseases for healthcare professionals serving local populations that live in endemic areas.

Tailored therapeutic release from polycaprolactone-silica hybrids for the treatment of osteomyelitis: antibiotic rifampicin and osteogenic silicates.

The treatment of osteomyelitis, a destructive inflammatory process caused by bacterial infections to bone tissue, is one of the most critical challenges of orthopedics and bone regenerative medicine. The standard treatment consists of intense antibiotic therapies combined with tissue surgical debridement and the application of a bone defect filler material. Unfortunately, commercially available candidates, such as gentamicin-impregnated polymethylmethacrylate cements, possess very poor pharmacokinetics (i.e., 24 hours burst release) and little to no regenerative potential. Fostered by the intrinsic limitations associated with conventional treatments, alternative osteostimulative biomaterials with local drug delivery have recently started to emerge. In this study, we propose the use of a polycaprolactone-silica sol-gel hybrid material as carrier for the delivery of rifampicin, an RNA-polymerase blocker often used to treat bone infections, and of osteostimulative silicate ions. The release of therapeutic agents from the material is dual, offering two separate and simultaneous effects, and decoupled, meaning that the kinetics of rifampicin and silicate releases are independent from each other. A series of hybrid formulations with increasing amounts of rifampicin was prepared. The antibiotic loading efficacy, as well as the release profiles of rifampicin and silicates were measured. The characterization of cell viability and differentiation of rat primary osteoblasts and antibacterial performance were also performed. Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa and Escherichia coli were selected due to their high occurrence in bone infections. Results confirmed that rifampicin can be successfully loaded within the hybrids without significant degradation and that it is possible to tailor the antibiotic release according to need. Once in a physiological environment, the rapid release of silicates was associated with optimal cell proliferation and the overexpression of osteoblastic differentiation. Simultaneously, rifampicin is delivered over the course of several weeks with significant inhibition of all tested strains. In particular, the materials caused a growth reduction of 7-10 orders of magnitude in Staphylococcus aureus, the major strain responsible for osteomyelitis worldwide. Our data strongly suggest that PCL/silica hybrids are a very promising candidate to develop bone fillers with superior biological performance compared to currently available options. Thanks to their unique synthesis route and their dual tailored release they can promote bone regeneration while reducing the risk of infection for several weeks upon implantation.

From Klebsiella pneumoniae Colonization to Dissemination: An Overview of Studies Implementing Murine Models.

Klebsiella pneumoniae is a Gram-negative pathogen responsible for community-acquired and nosocomial infections. The strains of this species belong to the opportunistic group, which is comprised of the multidrug-resistant strains, or the hypervirulent group, depending on their accessory genome, which determines bacterial pathogenicity and the host immune response. The aim of this survey is to present an overview of the murine models mimicking K. pneumoniae infectious processes (i.e., gastrointestinal colonization, urinary, pulmonary, and systemic infections), and the bacterial functions deployed to colonize and disseminate into the host. These in vivo approaches are pivotal to develop new therapeutics to limit K. pneumoniae infections via a modulation of the immune responses and/or microbiota.

Copper-Doped Biphasic Calcium Phosphate Powders: Dopant Release, Cytotoxicity and Antibacterial Properties.

Cytotoxicity and antibacterial properties associated with the dopant release of Cu-doped Biphasic Calcium Phosphate (BCP) powders, mainly composed of hydroxyapatite mixed with ß-tricalcium phosphate powders, were investigated. Twelve BCP ceramics were synthesized at three different sintering temperatures (600 °C, 900 °C and 1200 °C) and four copper doping rates (x = 0.0, 0.05, 0.10 and 0.20, corresponding to the stoichiometric amount of copper in Ca10Cux(PO4)6(OH)2-2xO2x). Cytotoxicity assessments of Cu-doped BCP powders, using MTT assay with human-Mesenchymal Stem Cells (h-MSCs), indicated no cytotoxicity and the release of less than 12 ppm of copper into the biological medium. The antibacterial activity of the powders was determined against both Gram-positive (methicillin-sensitive (MS) and methicillin resistant (MR) Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The Cu-doped biomaterials exhibited a strong antibacterial activity against MSSA, MRSA and E. coli, releasing approximatively 2.5 ppm after 24 h, whereas 10 ppm were required to induce an antibacterial effect against P. aeruginosa. This study also demonstrated that the culture medium used during experiments can directly impact the antibacterial effect observed; only 4 ppm of Cu2+ were effective for killing all the bacteria in a 1:500 diluted TS medium, whereas 20 ppm were necessary to achieve the same result in a rich, non-diluted standard marrow cell culture medium.

A prospective study on the pathogenesis of catheter-associated bacteriuria in critically ill patients.

BACKGROUND: Updating the pathogenesis of catheter-associated bacteriuria (CA-bacteriuria) in the intensive care unit (ICU) is needed to adapt prevention strategies. Our aim was to determine whether the main pathway of CA-bacteriuria in ICU patients was endoluminal or exoluminal. In a prospective study, quantitative urine cultures were sampled from catheter sampling sites, collector bags and the catheter outer surface near the meatus from days 1 to 15 after catheterization. The endoluminal pathway was CA-bacteriuria (defined as 102 CFU/mL) first in collector bags and then in catheters. The exoluminal pathway was CA-bacteriuria first in catheters, on day 1 in early cases and after day 1 in late cases. RESULTS: Of 64 included patients, 20 had CA-bacteriuria. Means of catheterization days and incidence density were 6.81 days and 55.2/1000 catheter-days. Of 26 microorganisms identified, 12 (46.2%) were Gram positive cocci, 8 (30.8%) Gram negative bacilli and 6 yeasts. Three (11.5%) CA-bacteriuria were endoluminal and 23 (88.5%) exoluminal, of which 10 (38.5%) were early and 13 (50%) late. Molecular comparison confirmed culture findings. A quality audit showed good compliance with guidelines. CONCLUSION: The exoluminal pathway of CA-bacteriuria in ICU patients predominated and surprisingly occurred early despite good implementation of guidelines. This finding should be considered in guidelines for prevention of CA-bacteriuria.

Biological properties of copper-doped biomaterials for orthopedic applications: A review of antibacterial, angiogenic and osteogenic aspects.

Copper is an essential trace element required for human life, and is involved in several physiological mechanisms. Today researchers have found and confirmed that Cu has biological properties which are particularly useful for orthopedic biomaterials applications such as implant coatings or biodegradable filler bone substitutes. Indeed, Cu exhibits antibacterial functions, provides angiogenic ability and favors osteogenesis; these represent major key points for ideal biomaterial integration and the healing process that follows. The antibacterial performances of copper-doped biomaterials present an interesting alternative to the massive use of prophylactic antibiotics and help to limit the development of antibiotic resistance. By stimulating blood vessel growth and new bone formation, copper contributes to the improved bio-integration of biomaterials. This review describes the bio-functional advantages offered by Cu and focuses on the antibacterial, angiogenic and osteogenic properties of Cu-doped biomaterials with potential for orthopedic applications.

Identification of sulfur components enhancing the anti-Candida effect of Lactobacillus rhamnosus Lcr35.

GYNOPHILUS (Lcr REGENERANS) is a live biotherapeutic product (LBP) aimed at restoring the vaginal microbiome and contains the live biotherapeutic microorganism Lactobacillus rhamnosus Lcr35. In this study, the LBP formulation and manufacturing process significantly enhanced the anti-Candida activity of L. rhamnosus Lcr35, with a complete loss of viability of the yeast after 48 h of coincubation. Sodium thiosulfate (STS), one excipient of the product, was used as a potentiator of the anti-Candida spp. activity of Lactobacilli. This contact-independent phenomenon induced fungal cell disturbances, as observed by electron microscopy observations. Nonverbal sensory experiments showed clear odor dissimilarities between cocultures of L. rhamnosus Lcr35 and C. albicans in the presence and absence of STS, suggesting an impact of odor-active metabolites. A volatolomic approach allowed the identification of six odor-active compounds, including one sulfur compound that was identified as S-methyl thioacetate (MTA). MTA was associated with the antifungal effect of Lcr35, and its functional link was established in vitro. We show for the first time that the LBP GYNOPHILUS, which is a highly active product in the reduction of vulvovaginal candidiasis, requires the presence of a sulfur compound to fully achieve its antifungal effect.

Fosfomycin and Staphylococcus aureus: transcriptomic approach to assess effect on biofilm, and fate of unattached cells.

Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

Antibiotic resilience: a necessary concept to complement antibiotic resistance?

Resilience is the capacity of systems to recover their initial state or functions after a disturbance. The concepts of resilience and resistance are complementary in ecology and both represent different aspects of the stability of ecosystems. However, antibiotic resilience is not used in clinical bacteriology whereas antibiotic resistance is a recognized major problem. To join the fields of ecology and clinical bacteriology, we first review the resilience concept from ecology, socio-ecological systems and microbiology where it is widely developed. We then review resilience-related concepts in microbiology, including bacterial tolerance and persistence, phenotypic heterogeneity and collective tolerance and resistance. We discuss how antibiotic resilience could be defined and argue that the use of this concept largely relies on its experimental measure and its clinical relevance. We review indicators in microbiology which could be used to reflect antibiotic resilience and used as valuable indicators to anticipate the capacity of bacteria to recover from antibiotic treatments.

Colonization and immune modulation properties of Klebsiella pneumoniae biofilm-dispersed cells.

Biofilm-dispersal is a key determinant for further dissemination of biofilm-embedded bacteria. Recent evidence indicates that biofilm-dispersed bacteria have transcriptional features different from those of both biofilm and planktonic bacteria. In this study, the in vitro and in vivo phenotypic properties of Klebsiella pneumoniae cells spontaneously dispersed from biofilm were compared with those of planktonic and sessile cells. Biofilm-dispersed cells, whose growth rate was the same as that of exponential planktonic bacteria but significantly higher than those of sessile and stationary planktonic forms, colonized both abiotic and biotic surfaces more efficiently than their planktonic counterparts regardless of their initial adhesion capabilities. Microscopy studies suggested that dispersed bacteria initiate formation of microcolonies more rapidly than planktonic bacteria. In addition, dispersed cells have both a higher engulfment rate and better survival/multiplication inside macrophages than planktonic cells and sessile cells. In an in vivo murine pneumonia model, the bacterial load in mice lungs infected with biofilm-dispersed bacteria was similar at 6, 24 and 48 h after infection to that of mice lungs infected with planktonic or sessile bacteria. However, biofilm-dispersed and sessile bacteria trend to elicit innate immune response in lungs to a lesser extent than planktonic bacteria. Collectively, the findings from this study suggest that the greater ability of K. pneumoniae biofilm-dispersed cells to efficiently achieve surface colonization and to subvert the host immune response confers them substantial advantages in the first steps of the infection process over planktonic bacteria.

Immunomodulatory Effects of Lactobacillus plantarum on Inflammatory Response Induced by Klebsiella pneumoniae.

Some respiratory infections have been associated with dysbiosis of the intestinal microbiota. The underlying mechanism is incompletely understood, but cross talk between the intestinal microbiota and local immune cells could influence the immune response at distal mucosal sites. This has led to the concept of enhancing respiratory defenses by modulating the intestinal microbiota with exogenous supplementation of beneficial strains. In this study, we examined the effect of Lactobacillus plantarum CIRM653 on the inflammatory response induced by the pathogen Klebsiella pneumoniae Oral administration of L. plantarum CIRM653 to mice subsequently infected by K. pneumoniae via the nasal route (i) reduced the pulmonary inflammation response, with decreased numbers of lung innate immune cells (macrophages and neutrophils) and cytokines (mouse keratinocyte-derived chemokine [KC], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) in the bronchoalveolar fluid, and (ii) induced an immunosuppressive Treg response in lungs. In vitro coincubation of L. plantarum CIRM653 and K. pneumoniae with human dendritic cells and peripheral blood mononuclear cells resulted in decreased Th1 (IL-12p70 and interferon gamma [IFN-γ]) and Th17 (IL-23 and IL-17) and increased Treg (IL-10) cytokine levels compared to those observed for K. pneumoniae-infected cells. Neither K. pneumoniae nor L. plantarum CIRM653 had any effect on cytokine production by intestinal epithelial cells in vitro, but the induction of the NF-κB pathway and IL-8 and IL-6 production by K. pneumoniae in airway epithelial cells was significantly reduced when the pathogen was coincubated with L. plantarum CIRM653. The remote IL-10-mediated modulation of the K. pneumoniae inflammatory response by L. plantarum CIRM653 supports the concept of immunomodulation by beneficial bacteria through the gut-lung axis.

Immobilisation of bacteria onto magnetic nanoparticles for the decolorisation and degradation of azo dyes.

Azo dyes are widely used in industries and their release in the environment contributes to the pollution of effluents. The authors aim to develop a new eco-friendly water treatment method for the degradation of azo dyes based on in situ magnetic separation and immobilisation of bacterial cells. The immobilisation was achieved using superparamagnetic Fe3O4 nanoparticles and offers the possibility of reusing bacteria by magnetic separation for several degradation cycles. The iron-oxide nanoparticles were synthesised by reverse co-precipitation. The Gram-positive bacteria Bacillus subtilis were immobilised using iron-oxide nanoparticles by adsorption and then separated with an external magnetic field. Transmission electron microscopy observation showed that the particles' diameter was ∼20 nm with a narrow size distribution. Moreover, the iron-oxide nanoparticles were adsorbed onto the surface in order to coat the cells. B. subtilis has proved its ability to decolorise and degrade several azo dyes at different values of pH, with the highest decolorisation rate for Congo red. Furthermore, immobilised cells have a degradation activity similar to that of free cells. The system provided a degradation rate up to 80% and could be reused for seven batch cycles.

1,2,3-Triazolium-Based Cationic Amphipathic Peptoid Oligomers Mimicking Antimicrobial Helical Peptides.

Amphipathic cationic peptoids (N-substituted glycine oligomers) represent a promising class of antimicrobial peptide mimics. The aim of this study is to explore the potential of the triazolium group as a cationic moiety and helix inducer to develop potent antimicrobial helical peptoids. Herein we report the first solid-phase synthesis of peptoid oligomers incorporating 1,2,3-triazolium-type side chains and their evaluation against Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. Several triazolium-based oligomers, even of short length, selectively kill bacteria over mammalian cells. SEM visualization of S. aureus cells treated with a dodecamer and a hexamer reveals severe cell membrane damage and suggests that the longer oligomer acts by pore formation.

How do environment-dependent switching rates between susceptible and persister cells affect the dynamics of biofilms faced with antibiotics?

Persisters form sub-populations of stress-tolerant cells that play a major role in the capacity of biofilms to survive and recover from disturbances such as antibiotic treatments. The mechanisms of persistence are diverse and influenced by environmental conditions, and persister populations are more heterogeneous than formerly suspected. We used computational modeling to assess the impact of three switching strategies between susceptible and persister cells on the capacity of bacterial biofilms to grow, survive and recover from antibiotic treatments. The strategies tested were: (1) constant switches, (2) substrate-dependent switches and (3) antibiotic-dependent switches. We implemented these strategies in an individual-based biofilm model and simulated antibiotic shocks on virtual biofilms. Because of limited available data on switching rates in the literature, nine parameter sets were assessed for each strategy. Substrate and antibiotic-dependent switches allowed high switching rates without affecting the growth of the biofilms. Compared to substrate-dependent switches, constant and antibiotic-dependent switches were associated with higher proportions of persisters in the top of the biofilms, close to the substrate source, which probably confers a competitive advantage within multi-species biofilms. The constant and substrate-dependent strategies need a compromise between limiting the wake-up and death of persisters during treatments and leaving the persister state fast enough to recover quickly after antibiotic-removal. Overall, the simulations gave new insights into the relationships between the dynamics of persister populations in biofilms and their dynamics of growth, survival and recovery when faced with disturbances.

Unveiling and Characterizing Early Bilateral Interactions between Biofilm and the Mouse Innate Immune System.

A very substantial progress has been made in our understanding of infectious diseases caused by invasive bacteria. Under their planktonic forms, bacteria transiently reside in the otherwise sterile mammal body tissues, as the physiological inflammation insures both their clearance and repair of any tissue damage. Yet, the bacteria prone to experience planktonic to biofilm developmental transition still need to be studied. Of note, sessile bacteria not only persist but also concur preventing the effectors and regulators of the physiological inflammation to operate. Thus, it is urgent to design biologically sound experimental approaches aimed to extract, at the earliest stage, immune signatures of mono-bacteria planktonic to biofilm developmental transition in vivo and ex vivo. Indeed, the transition is often the first event to which succeeds the "chronicization" process whereby classical bacteria-targeting therapies are no more efficacious. An in vivo model of micro-injection of Staphylococcus aureus planktonic or biofilm cells in the ear pinna dermis of laboratory transgenic mice with fluorescent immune cells is proposed. It allows visualizing, in real time, the range of the early interactions between the S. aureus and myeloid cell subsets- the resident macrophages and dendritic cells, the recruited neutrophil granulocytes/polymorphonuclear neutrophils, monocytes otherwise known to differentiate as macrophages or dendritic cells. One main objective is to extract contrasting immune signatures of the modulation of the physiological inflammation with respect to the two bacterial lifestyles.