Bacteroides thetaiotaomicron uses a widespread extracellular DNase to promote bile-dependent biofilm formation.

Bacteroides thetaiotaomicron is a gut symbiont that inhabits the mucus layer and adheres to and metabolizes food particles, contributing to gut physiology and maturation. Although adhesion and biofilm formation could be key features for B. thetaiotaomicron stress resistance and gut colonization, little is known about the determinants of B. thetaiotaomicron biofilm formation. We previously showed that the B. thetaiotaomicron reference strain VPI-5482 is a poor in vitro biofilm former. Here, we demonstrated that bile, a gut-relevant environmental cue, triggers the formation of biofilm in many B. thetaiotaomicron isolates and common gut Bacteroidales species. We determined that bile-dependent biofilm formation involves the production of the DNase BT3563 or its homologs, degrading extracellular DNA (eDNA) in several B. thetaiotaomicron strains. Our study therefore shows that, although biofilm matrix eDNA provides a biofilm-promoting scaffold in many studied Firmicutes and Proteobacteria, BT3563-mediated eDNA degradation is required to form B. thetaiotaomicron biofilm in the presence of bile.

Insights into the genomic and phenotypic characteristics of Bacillus spp. strains isolated from biofilms in broiler farms.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

Bacterial adhesion inhibition by microalgal EPSs from Cylindrotheca closterium and Tetraselmis suecica biofilms.

In the food industry, successful bacterial pathogen colonization and persistence begin with their adhesion to a surface, followed by the spatial development of mature biofilm of public health concerns. Compromising bacterial settlement with natural inhibitors is a promising alternative to conventional anti-fouling treatments typically based on chemical biocides that contribute to the growing burden of antimicrobial resistance. In this study, three extracellular polymeric substance (EPS) fractions extracted from microalgae biofilms of Cylindrotheca closterium (fraction C) and Tetraselmis suecica (fraction Ta rich in insoluble scale structure and fraction Tb rich in soluble EPS) were screened for their anti-adhesive properties, against eight human food-borne pathogens belonging to Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterica, and Listeria monocytogenes species. The results showed that the fraction Ta was the most effective inducing statistically significant reduction for three strains of E. coli, S. aureus, and L. monocytogenes. Overall, EPSs coating on polystyrene surfaces of the different fractions increased the hydrophilic character of the support. Differences in bacterial adhesion on the different coated surfaces could be explained by several dissimilarities in the structural and physicochemical EPS compositions, according to HPLC and ATR-FTIR analysis. Interestingly, while fractions Ta and Tb were extracted from the same microalgal culture, distinct adhesion patterns were observed, highlighting the importance of the extraction process. Overall, the findings showed that EPS extracted from microalgal photosynthetic biofilms can exhibit anti-adhesive effects against food-borne pathogens and could help develop sustainable and non-toxic anti-adhesive surfaces for the food industry. KEY POINTS: •EPSs from a biofilm-based culture of C. closterium/T. suecica were characterized. •Microalgal EPS extracted from T. suecica biofilms showed bacterial anti-adhesive effects. •The anti-adhesive effect is strain-specific and affects both Gram - and Gram + bacteria.

Biofilm formation of the food spoiler Brochothrix thermosphacta on different industrial surface materials using a biofilm reactor.

Brochothrix thermosphacta is considered as a major food spoiler bacteria. This study evaluates biofilm formation by B. thermosphacta CD337(2) - a strong biofilm producer strain - on three food industry materials (polycarbonate (PC), polystyrene (PS), and stainless steel (SS)). Biofilms were continuously grown under flow at 25 °C in BHI broth in a modified CDC biofilm reactor. Bacterial cells were enumerated by plate counting, and biofilm spatial organization was deciphered by combining confocal laser scanning microscopy and image analysis. The biofilms had the same growth kinetics on all three materials and reach 8log CFU/cm2 as maximal concentration. Highly structured biofilms were observed on PC and PS, but less structured ones on SS. This difference was confirmed by structural quantification analysis using the image analysis software tool BiofilmQ. Biofilm on SS show less roughness, density, thickness and volume. The biofilm 3D structure seemed to be related to the coupon topography and roughness. The materials used in this study do not affect biofilm growth. However, their roughness and topography affect the biofilm architecture, which could influence biofilm behaviour.

Biofilms as protective cocoons against biocides: from bacterial adaptation to One Health issues.

Bacteria in the food chain mostly live in communities associated with surfaces known as biofilms, which confer specific survival and adaptive abilities. In such communities, the bacteria mostly exhibit higher tolerance to external stress, and their recurrent exposure along the food chain to biocides used during cleaning and disinfection procedures raises concern about the adaptation routes they develop, both at single-cell and communal levels. In recent years, an increasing number of research subjects have focused on understanding the specific features of biofilms that enable bacterial populations to adapt to biocide exposure within a 'protective cocoon'. The first part of this review concentrates on the diversity of adaptive strategies, including structural modulation of these biofilms, physiological response or the acquisition of genetic resistance. The second part discusses the possible side effects of biofilm adaptation to biocides on antimicrobial cross-resistance, virulence and colonization features from a One Health perspective.

The architecture and metabolic traits of monospecific photosynthetic biofilms studied in a custom flow-through system.

Microalgae biofilms have great ecological importance and high biotechnological potential. Nevertheless, an in-depth and combined structural (i.e., the architecture of the biofilm) and physiological characterization of microalgae biofilms is still missing. An approach able to provide the same time physiological and structural information during biofilm growth would be of paramount importance to understand these complex biological systems and to optimize their productivity. In this study, monospecific biofilms of a diatom and a green alga were grown under dynamic conditions in custom flow cells represented by UV/Vis spectroscopic cuvettes. Such flow cells were conceived to characterize the biofilms by several techniques mostly in situ and in a nondestructive way. Physiological traits were obtained by measuring variable chlorophyll a fluorescence by pulse amplitude modulated fluorometry and by scanning the biofilms in a spectrometer to obtain in vivo pigments spectral signatures. The architectural features were obtained by imaging the biofilms with a confocal laser scanning microscopy and an optical coherence tomography. Overall, this experimental setup allowed us to follow the growth of two biofilm-forming microalgae showing that cell physiology is more affected in complex biofilms likely as a consequence of alterations in local environmental conditions.

Spatial organisation of Listeria monocytogenes and Escherichia coli O157:H7 cultivated in gel matrices.

The spatial organisation of bacterial pathogens in food matrices remains poorly understood, but is important in improving risk assessment and preventing infection of consumers by contaminated foodstuff. By combining confocal laser scanning microscopy with genetic fluorescent labelling of Listeria monocytogenes and Escherichia coli O157:H7, it was possible to investigate the spatial patterns of colonisation of both foodborne pathogens in gel matrices, alone or in combination, in various environmental conditions. Increasing low melting point agarose (LMPA) concentrations triggers the transition between a motile single-cell lifestyle to a sessile population spatially organised as microcolonies. The size, number and morphology of microcolonies were highly affected by supplementations in NaCl or lactic acid, two compounds frequently used in food products. Strikingly, single-cell motility was partially restored at higher LMPA concentration in the presence of lactic acid for Escherichia coli O157:H7 and in the presence of NaCl for Listeria monocytogenes. Co-culture of both species in the hydrogel affected pathogen colonisation features; Listeria monocytogenes was better able to colonise gel matrices containing lactic acid in the presence of Escherichia coli O157:H7. Altogether, this investigation provides insights into the spatial distribution and structural dynamics of bacterial pathogens in gel matrices. Potential impacts on food safety are discussed.

Genetic, physiological, and cellular heterogeneities of bacterial pathogens in food matrices: Consequences for food safety.

In complex food systems, bacteria live in heterogeneous microstructures, and the population displays phenotypic heterogeneities at the single-cell level. This review provides an overview of spatiotemporal drivers of phenotypic heterogeneity of bacterial pathogens in food matrices at three levels. The first level is the genotypic heterogeneity due to the possibility for various strains of a given species to contaminate food, each of them having specific genetic features. Then, physiological heterogeneities are induced within the same strain, due to specific microenvironments and heterogeneous adaptative responses to the food microstructure. The third level of phenotypic heterogeneity is related to cellular heterogeneity of the same strain in a specific microenvironment. Finally, we consider how these phenotypic heterogeneities at the single-cell level could be implemented in mathematical models to predict bacterial behavior and help ensure microbiological food safety.

Biofilm Formation and Synthesis of Antimicrobial Compounds by the Biocontrol Agent Bacillus velezensis QST713 in an Agaricus bisporus Compost Micromodel.

Bacillus velezensis QST713 is widely used as a biological control agent for crop protection and disease suppression. This strain is used industrially in France for the protection of Agaricus bisporus against Trichoderma aggressivum f. europaeum, which causes green mold disease. The efficacy of this biocontrol process was evaluated in a previous study, yet the mode of its action has not been explored under production conditions. In order to decipher the underlying biocontrol mechanisms for effective biofilm formation by strain QST713 in the compost and for the involvement of antimicrobial compounds, we developed a simplified micromodel for the culture of A. bisporus during its early culture cycle. By using this micromodel system, we studied the transcriptional response of strain QST713 in the presence or absence of A. bisporus and/or T. aggressivum in axenic industrial compost. We report the overexpression of several genes of the biocontrol agent involved in biofilm formation in the compost compared to their expression during growth in broth compost extract either in the exponential growth phase (the epsC, blsA, and tapA genes) or in the stationary growth phase (the tapA gene), while a gene encoding a flagellar protein (hag) was underexpressed. We also report the overexpression of Bacillus velezensis QST713 genes related to surfactin (srfAA) and fengycin (fenA) production in the presence of the fungal pathogen in the compost.IMPORTANCE Biocontrol agents are increasingly used to replace chemical pesticides to prevent crop diseases. In the button mushroom field in France, the use of Bacillus velezensis QST713 as a biocontrol agent against the green mold Trichoderma aggressivum has been shown to be efficient. However, the biocontrol mechanisms effective in the Agaricus bisporus/Trichoderma aggressivum/Bacillus velezensis QST713 pathosystem are still unknown. Our paper focuses on the exploration of the bioprotection mechanisms of the biocontrol agent Bacillus velezensis QST713 during culture of the button mushroom (Agaricus bisporus) in a micromodel culture system to study the specific response of strain QST713 in the presence of T. aggressivum and/or A. bisporus.

Blp1 protein shows virulence-associated features and elicits protective immunity to Acinetobacter baumannii infection.

BACKGROUND: Multidrug resistant Acinetobacter baumannii is one of the major infection agents causing nosocomial pneumonia. Therefore, new therapeutic approaches against this bacterium are needed. Surface-exposed proteins from bacterial pathogens are implicated in a variety of virulence-related traits and are considered as promising candidates for vaccine development. RESULTS: We show in this study that a large Blp1 protein from opportunistic pathogen A. baumannii is encoded in all examined clinical strains of globally spread international clonal lineages I (IC I) and II (IC II). The two blp1 gene variants exhibit lineage-specific distribution profile. By characterization of blp1 deletion mutants and their complementation with blp1 alleles we show that blp1 gene is required for A. baumannii biofilm formation and adhesion to epithelial cells in IC I strain but not in the IC II strain. Nevertheless both alleles are functional in restoring the deficient phenotypes of IC I strain. Moreover, the blp1 gene is required for the establishing of A. baumannii virulence phenotype in nematode and murine infection models. Additionally, we demonstrate that C-terminal 711 amino acid fragment of Blp1 elicits an efficient protection to lethal A. baumannii infection in a murine model using active and passive immunization approaches. Antiserum obtained against Blp1-specific antigen provides opsonophagocytic killing of A. baumannii in vitro. CONCLUSIONS: Lineage-specific variants of surface-exposed components of bacterial pathogens complicate the development of new therapeutic approaches. Though we demonstrated different impact of Blp1 variants on adherence of IC I and IC II strains, Blp1-specific antiserum neutralized A. baumannii strains of both clonal lineages. Together with the observed increased survival rate in vaccinated mice these results indicate that A. baumannii Blp1 protein could be considered as a new vaccine candidate.

The Mutation of Conservative Asp268 Residue in the Peptidoglycan-Associated Domain of the OmpA Protein Affects Multiple Acinetobacter baumannii Virulence Characteristics.

Acinetobacter baumannii is a nosocomial human pathogen of increasing concern due to its multidrug resistance profile. The outer membrane protein A (OmpA) is an abundant bacterial cell surface component involved in A. baumannii pathogenesis. It has been shown that the C-terminal domain of OmpA is located in the periplasm and non-covalently associates with the peptidoglycan layer via two conserved amino acids, thereby anchoring OmpA to the cell wall. Here, we investigated the role of one of the respective residues, D268 in OmpA of A. baumannii clinical strain Ab169, on its virulence characteristics by complementing the ΔompA mutant with the plasmid-borne ompAD268A allele. We show that while restoring the impaired biofilm formation of the ΔompA strain, the Ab169ompAD268A mutant tended to form bacterial filaments, indicating the abnormalities in cell division. Moreover, the Ab169 OmpA D268-mediated association to peptidoglycan was required for the manifestation of twitching motility, desiccation resistance, serum-induced killing, adhesion to epithelial cells and virulence in a nematode infection model, although it was dispensable for the uptake of ß-lactam antibiotics by outer membrane vesicles. Overall, the results of this study demonstrate that the OmpA C-terminal domain-mediated association to peptidoglycan is critical for a number of virulent properties displayed by A. baumannii outside and within the host.

Impact of Bacterial Membrane Fatty Acid Composition on the Failure of Daptomycin To Kill Staphylococcus aureus.

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 against Staphylococcus 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 against S. aureus varies 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 treat S. aureus is 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 restore S. aureus daptomycin bactericidal activity in vivo could open the way to novel antibiotic treatment strategies.

Live intramacrophagic Staphylococcus aureus as a potential cause of antibiotic therapy failure: observations in an in vivo mouse model of prosthetic vascular material infections.

Objectives: To evaluate the significant role played by biofilms during prosthetic vascular material infections (PVMIs). Methods: We developed an in vivo mouse model of Staphylococcus aureus PVMI allowing its direct observation by confocal microscopy to describe: (i) the structure of biofilms developed on Dacron® vascular material; (ii) the localization and effect of antibiotics on these biostructures; and (iii) the interaction between bacteria and host tissues and cells during PVMI. Results: In this model we demonstrated that the biofilm structures are correlated to the activity of antibiotics. Furthermore, live S. aureus bacteria were visualized inside the macrophages present at the biofilm sites, which is significant as antibiotics do not penetrate these immune cells. Conclusions: This intracellular situation may explain the limited effect of antibiotics and also why PVMIs can relapse after antibiotic therapy.

Impact of Cell Surface Molecules on Conjugative Transfer of the Integrative and Conjugative Element ICESt3 of Streptococcus thermophilus.

Integrative conjugative elements (ICEs) are chromosomal elements that are widely distributed in bacterial genomes, hence contributing to genome plasticity, adaptation, and evolution of bacteria. Conjugation requires a contact between both the donor and the recipient cells and thus likely depends on the composition of the cell surface envelope. In this work, we investigated the impact of different cell surface molecules, including cell surface proteins, wall teichoic acids, lipoteichoic acids, and exopolysaccharides, on the transfer and acquisition of ICESt3 from Streptococcus thermophilus The transfer of ICESt3 from wild-type (WT) donor cells to mutated recipient cells increased 5- to 400-fold when recipient cells were affected in lipoproteins, teichoic acids, or exopolysaccharides compared to when the recipient cells were WT. These mutants displayed an increased biofilm-forming ability compared to the WT, suggesting better cell interactions that could contribute to the increase of ICESt3 acquisition. Microscopic observations of S. thermophilus cell surface mutants showed different phenotypes (aggregation in particular) that can also have an impact on conjugation. In contrast, the same mutations did not have the same impact when the donor cells, instead of recipient cells, were mutated. In that case, the transfer frequency of ICESt3 decreased compared to that with the WT. The same observation was made when both donor and recipient cells were mutated. The dominant effect of mutations in donor cells suggests that modifications of the cell envelope could impair the establishment or activity of the conjugation machinery required for DNA transport.IMPORTANCE ICEs contribute to horizontal gene transfer of adaptive traits (for example, virulence, antibiotic resistance, or biofilm formation) and play a considerable role in bacterial genome evolution, thus underlining the need of a better understanding of their conjugative mechanism of transfer. While most studies focus on the different functions encoded by ICEs, little is known about the effect of host factors on their conjugative transfer. Using ICESt3 of S. thermophilus as a model, we demonstrated the impact of lipoproteins, teichoic acids, and exopolysaccharides on ICE transfer and acquisition. This opens up new avenues to control gene transfer mediated by ICEs.

Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine-rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram-positive bacterial genomes have a unique SRR glycoprotein-encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface-exposed proteins - SrpA, SrpB and SrpC - that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases - GtfE/F - encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto-aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.

Impact of temperature on Marinobacter hydrocarbonoclasticus SP17 morphology and biofilm structure during growth on alkanes.

Alkanes are widespread pollutants found in soil, freshwater and marine environments. Marinobacter hydrocarbonoclasticus (Mh) strain SP17 is a marine bacterium able to use many hydrophobic organic compounds, including alkanes, through the production of biofilms that allow their poor solubility to be overcome. This study pointed out that temperature is an environmental factor that strongly affects the biofilm formation and morphology of Mh on the model alkanes, hexadecane and paraffin. We showed that Mh biofilm formation and accumulation of intracytoplasmic inclusions are higher on solid alkanes (hexadecane at 10 °C and paraffin at 10 °C and 30 °C) than on liquid alkane (hexadecane at 30 °C) or soluble substrate (lactate at both temperatures). We also found that Mh produces more extracellular polymeric substances at 30 °C than at 10 °C on alkanes and none on lactate. We observed that bacterial length is significantly higher at 10 °C than at 30 °C on lactate and hexadecane. On paraffin, at 30 °C, the cell morphology is markedly altered by large rounded or irregularly shaped cytoplasmic inclusions. Altogether, the results showed that Mh is able to adapt and use alkanes as a carbon source, even at low temperature.

Cleaning and Disinfection of Biofilms Composed of Listeria monocytogenes and Background Microbiota from Meat Processing Surfaces.

Surfaces of food processing premises are exposed to regular cleaning and disinfection (C&D) regimes, using biocides that are highly effective against bacteria growing as planktonic cells. However, bacteria growing in surface-associated communities (biofilms) are typically more tolerant toward C&D than their individual free-cell counterparts, and survival of pathogens such as Listeria monocytogenes may be affected by interspecies interactions within biofilms. In this study, Pseudomonas and Acinetobacter were the most frequently isolated genera surviving on conveyor belts subjected to C&D in meat processing plants. In the laboratory, Pseudomonas, Acinetobacter, and L. monocytogenes dominated the community, both in suspensions and in biofilms formed on conveyor belts, when cultures were inoculated with eleven-genus cocktails of representative bacterial strains from the identified background flora. When biofilms were exposed to daily C&D cycles mimicking treatments used in food industry, the levels of Acinetobacter and Pseudomonas mandelii diminished, and biofilms were instead dominated by Pseudomonas putida (65 to 76%), Pseudomonas fluorescens (11 to 15%) and L. monocytogenes (3 to 11%). The dominance of certain species after daily C&D correlated with high planktonic growth rates at 12°C and tolerance to C&D. In single-species biofilms, L. monocytogenes developed higher tolerance to C&D over time, for both the peracetic acid and quaternary ammonium disinfectants, indicating that a broad-spectrum mechanism was involved. Survival after C&D appeared to be a common property of L. monocytogenes strains, as persistent and sporadic subtypes showed equal survival rates in complex biofilms. Biofilms established preferentially in surface irregularities of conveyor belts, potentially constituting harborage sites for persistent contamination.IMPORTANCE In the food industry, efficient production hygiene is a key measure to avoid the accumulation of spoilage bacteria and eliminate pathogens. However, the persistence of bacteria is an enduring problem in food processing environments. This study demonstrated that environmental bacteria can survive foam cleaning and disinfection (C&D) at concentrations used in the industrial environment. The phenomenon was replicated in laboratory experiments. Important characteristics of persisting bacteria were a high growth rate at low temperature, a tolerance to the cleaning agent, and the ability to form biofilms. This study also supports other recent research suggesting that strain-to-strain variation cannot explain why certain subtypes of Listeria monocytogenes persist in food processing environments while others are found only sporadically. The present investigation highlights the failure of regular C&D and a need for research on improved agents that efficiently detach the biofilm matrix.

Critical review on biofilm methods.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

How do fluorescence spectroscopy and multimodal fluorescence imaging help to dissect the enhanced efficiency of the vancomycin-rifampin combination against Staphylococcus aureus infections?

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.

New Insight into Daptomycin Bioavailability and Localization in Staphylococcus aureus Biofilms by Dynamic Fluorescence Imaging.

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.