Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm

Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilm-associated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone.     

Differential Roles of Poly-N-Acetylglucosamine Surface Polysaccharide and Extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis Biofilms

Staphylococcus aureus and Staphylococcus epidermidis are major human pathogens of increasing importance due to the dissemination of antibiotic-resistant strains. Evidence suggests that the ability to form matrix-encased biofilms contributes to the pathogenesis of S. aureus and S. epidermidis. In this study, we investigated the functions of two staphylococcal biofilm matrix polymers: poly-N-acetylglucosamine surface polysaccharide (PNAG) and extracellular DNA (ecDNA). We measured the ability of a PNAG-degrading enzyme (dispersin B) and DNase I to inhibit biofilm formation, detach preformed biofilms, and sensitize biofilms to killing by the cationic detergent cetylpyridinium chloride (CPC) in a 96-well microtiter plate assay. When added to growth medium, both dispersin B and DNase I inhibited biofilm formation by both S. aureus and S. epidermidis. Dispersin B detached preformed S. epidermidis biofilms but not S. aureus biofilms, whereas DNase I detached S. aureus biofilms but not S. epidermidis biofilms. Similarly, dispersin B sensitized S. epidermidis biofilms to CPC killing, whereas DNase I sensitized S. aureus biofilms to CPC killing. We concluded that PNAG and ecDNA play fundamentally different structural roles in S. aureus and S. epidermidis biofilms.     

Surface Acoustic Waves Enhance Neutrophil Killing of Bacteria

Biofilms are structured communities of bacteria that play a major role in the pathogenicity of bacteria and are the leading cause of antibiotic resistant bacterial infections on indwelling catheters and medical prosthetic devices. Failure to resolve these biofilm infections may necessitate the surgical removal of the prosthetic device which can be debilitating and costly. Recent studies have shown that application of surface acoustic waves to catheter surfaces can reduce the incidence of infections by a mechanism that has not yet been clarified. We report here the effects of surface acoustic waves (SAW) on the capacity of human neutrophils to eradicate S. epidermidis bacteria in a planktonic state and within biofilms. Utilizing a novel fibrin gel system that mimics a tissue-like environment, we show that SAW, at an intensity of 0.3 mW/cm², significantly enhances human neutrophil killing of S. epidermidis in a planktonic state and within biofilms by enhancing human neutrophil chemotaxis in response to chemoattractants. In addition, we show that the integrin CD18 plays a significant role in the killing enhancement observed in applying SAW. We propose from out data that this integrin may serve as mechanoreceptor for surface acoustic waves enhancing neutrophil chemotaxis and killing of bacteria.     

Genetic Diversity of the Biofilm Covering Montacuta ferruginosa (Mollusca, Bivalvia) as Evaluated by Denaturing Gradient Gel Electrophoresis Analysis and Cloning of PCR-Amplified Gene Fragments Coding for 16S rRNA

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the γ-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.     

Phenotypic and genotypic characterization of Staphylococci causing breast peri-implant infections in oncologic patients

Background

Staphylococcus epidermidis and S. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. Biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. To our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections.

Results

Thirteen S. aureus and seven S. epidermidis causing breast peri-implant infections were studied.Using the broth microdilution method and the E-test, the majority of the strains were susceptible to all antibiotics tested. Methicillin resistance was detected in two S. epidermidis. All strains had different RAPD profiles and were able to produce biofilms in microtitre plate assays but, while all S. aureus carried and were able to express icaA and icaD genes, this was only true for one S. epidermidis. Biofilm development was glucose- and NaCl-induced (5 S. aureus and 1 S. epidermidis) or glucose-induced (the remaining strains). Proteinase K and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation.All S. aureus strains harboured and expressed the atlA, clfA, FnA, eno and cna genes and the majority also carried and expressed the sasG (10/13), ebpS (10/13) genes.All S. epidermidis strains harboured and expressed the atlE, aae, embp genes, and the majority (six strains) also carried and expressed the fbe, aap genes.Genes for S. aureus capsular types 5 and 8 were almost equally distributed. The only leukotoxin genes detected were lukE/lukD (6/13).

Conclusions

S. aureus and S. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms.Since the collagen adhesin (cna) gene is not ubiquitously distributed among S. aureus, this protein could have an important role in the cause of breast peri-implant infections.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0368-x) contains supplementary material, which is available to authorized users.     

The Small Molecule DAM Inhibitor,Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.     

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Background

Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.

Materials and Methods

Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.

Results

No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.

Conclusion

Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.     

Encapsulation in Polymeric Microparticles Improves Daptomycin Activity Against Mature Staphylococci Biofilms—a Thermal and Imaging Study

Eradication of Gram-positive biofilms is a critical aspect in implant-associated infection treatment. Although antibiotic-containing particulate carriers may be a promising strategy for overcoming biofilm tolerance, the assessment of their interaction with biofilms has not been fully explored. In the present work, the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL 100 (EUD) microparticles against methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive S. epidermidis biofilms was investigated using isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH). The minimal biofilm inhibitory concentrations (MBIC) of MRSA biofilms, as determined by IMC, were 5 and 20 mg/mL for daptomycin- and vancomycin-loaded PMMA microparticles, respectively. S. epidermidis biofilms were less susceptible, with a MBIC of 20 mg/mL for daptomycin-loaded PMMA microparticles. Vancomycin-loaded microparticles were ineffective. Adding EUD to the formulation caused a 4- and 16-fold reduction of the MBIC values of daptomycin-loaded microparticles for S. aureus and S. epidermidis, respectively. FISH corroborated the IMC results and provided additional insights on the antibiofilm effect of these particles. According to microscopic analysis, only daptomycin-loaded PMMA-EUD microparticles were causing a pronounced reduction in biofilm mass for both strains. Taken together, although IMC indicated that a biofilm inhibition was achieved, microscopy showed that the biofilm was not eradicated and still contained FISH-positive, presumably viable bacteria, thus indicating that combining the two techniques is essential to fully assess the effect of microparticles on staphylococcal biofilms.     

In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci

 

Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown.

Methods

We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells.

Results

Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 μg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis

Conclusion

The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.     

Antibiotic-resistant Staphylococcus epidermidis isolated from patients and healthy students comparing with antibiotic-resistant bacteria isolated from pasteurized milk

Antibiotic-resistant Staphylococci are a global issue affecting humans, animals, and numerous natural environments. Antibiotic-resistant Staphylococcus epidermidis is an opportunistic pathogen frequently isolated from patients and healthy individuals. This study aimed to examine the antibiotic resistance of S. epidermidis isolated from patients, healthy students and compare the results with antibiotic-resistant bacteria isolated from pasteurized milk. Clinical strain isolation was performed in several hospitals in the Riyadh. Skin swabs from 100 healthy undergraduate candidate students were obtained at King Saud University. The pasteurized milk samples were obtained from local market (company, X). After isolation, identification and susceptibility tests were performed using an automated system. A multiplex tuf gene-based PCR assay was used to confirm identification. Biofilm production and biofilm-related gene expression were studied. S. epidermidis represented 17% of clinical bacterial isolates, and 1.7% of isolates obtained from healthy students were multiantibiotic-resistant. All patient strains were teicoplanin- and vancomycin-susceptible, while all student strains were gentamicin-, levofloxacin-, moxifloxacin-, and trimethoprim/sulfamethoxazole-susceptible. All the bacteria isolated from pasteurized milk were benzylpenicillin and oxacillin-resistant strains. Of the S. epidermidis strains, 91% could produce biofilms, and mecA, icaADBR, ica-ADB, ica-AD, ica-A only, and ica-C only were expressed in 83, 17.1, 25.7, 37.1, 20, and 0% of the strains, respectively. This work demonstrates that S. epidermidis can be accurately identified using a multiplex tuf-based assay, and that multiantibiotic-resistant S. epidermidis strains are widespread amongst patients and healthy students.     

Biofilm Morphotypes and Population Structure among Staphylococcus epidermidis from Commensal and Clinical Samples

Bacterial species comprise related genotypes that can display divergent phenotypes with important clinical implications. Staphylococcus epidermidis is a common cause of nosocomial infections and, critical to its pathogenesis, is its ability to adhere and form biofilms on surfaces, thereby moderating the effect of the host’s immune response and antibiotics. Commensal S. epidermidis populations are thought to differ from those associated with disease in factors involved in adhesion and biofilm accumulation. We quantified the differences in biofilm formation in 98 S. epidermidis isolates from various sources, and investigated population structure based on ribosomal multilocus typing (rMLST) and the presence/absence of genes involved in adhesion and biofilm formation. All isolates were able to adhere and form biofilms in in vitro growth assays and confocal microscopy allowed classification into 5 biofilm morphotypes based on their thickness, biovolume and roughness. Phylogenetic reconstruction grouped isolates into three separate clades, with the isolates in the main disease associated clade displaying diversity in morphotype. Of the biofilm morphology characteristics, only biofilm thickness had a significant association with clade distribution. The distribution of some known adhesion-associated genes (aap and sesE) among isolates showed a significant association with the species clonal frame. These data challenge the assumption that biofilm-associated genes, such as those on the ica operon, are genetic markers for less invasive S. epidermidis isolates, and suggest that phenotypic characteristics, such as adhesion and biofilm formation, are not fixed by clonal descent but are influenced by the presence of various genes that are mobile among lineages.     

Anti-biofilm properties of the antimicrobial peptide temporin 1Tb and its ability,in combination with EDTA,to eradicate Staphylococcus epidermidis biofilms on silicone catheters

In search of new antimicrobials with anti-biofilm potential, in the present study activity of the frog-skin derived antimicrobial peptide temporin 1Tb (TB) against Staphylococcus epidermidis biofilms was investigated. A striking ability of TB to kill both forming and mature S. epidermidis biofilms was observed, especially when the peptide was combined with cysteine or EDTA, respectively. Kinetics studies demonstrated that the combination TB/EDTA was active against mature biofilms already after 2–4-h exposure. A double 4-h exposure of biofilms to TB/EDTA further increased the therapeutic potential of the same combination. Of note, TB/EDTA was able to eradicate S. epidermidis biofilms formed in vitro on silicone catheters. At eradicating concentrations, TB/EDTA did not cause hemolysis of human erythrocytes. The results shed light on the anti-biofilm properties of TB and suggest a possible application of the peptide in the lock therapy of catheters infected with S. epidermidis.     

Influence of fluoride on the bacterial composition of a dual-species biofilm composed of Streptococcus mutans and Streptococcus oralis

Despite the widespread use of fluoride for the prevention of dental caries, few studies have demonstrated the effects of fluoride on the bacterial composition of dental biofilms. This study investigated whether fluoride affects the proportion of Streptococcus mutans and S. oralis in mono- and dual-species biofilm models, via microbiological, biochemical, and confocal fluorescence microscope studies. Fluoride did not affect the bacterial count and bio-volume of S. mutans and S. oralis in mono-species biofilms, except for the 24-h-old S. mutans biofilms. However, fluoride reduced the proportion and bio-volume of S. mutans but did not decrease those of S. oralis during both S. oralis and S. mutans dual-species biofilm formation, which may be related to the decrease in extracellular polysaccharide formation by fluoride. These results suggest that fluoride may prevent the shift in the microbial proportion to cariogenic bacteria in dental biofilms, subsequently inhibiting the cariogenic bacteria dominant biofilm formation.     

Effects of Growth in the Presence of Subinhibitory Concentrations of Dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus Biofilms

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions ofbiofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.     

Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Staphylococcus aureus and Staphylococcus epidermidis Virulence Strains as Causative Agents of Persistent Infections in Breast Implants

Staphylococcus epidermidis and Staphylococcus aureus are currently considered two of the most important pathogens in nosocomial infections associated with catheters and other medical implants and are also the main contaminants of medical instruments. However because these species of Staphylococcus are part of the normal bacterial flora of human skin and mucosal surfaces, it is difficult to discern when a microbial isolate is the cause of infection or is detected on samples as a consequence of contamination. Rapid identification of invasive strains of Staphylococcus infections is crucial for correctly diagnosing and treating infections. The aim of the present study was to identify specific genes to distinguish between invasive and contaminating S. epidermidis and S. aureus strains isolated on medical devices; the majority of our samples were collected from breast prostheses. As a first step, we compared the adhesion ability of these samples with their efficacy in forming biofilms; second, we explored whether it is possible to determine if isolated pathogens were more virulent compared with international controls. In addition, this work may provide additional information on these pathogens, which are traditionally considered harmful bacteria in humans, and may increase our knowledge of virulence factors for these types of infections.     

An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.     

Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other organisms. Our data provide insights about how S. mutans optimizes its metabolism and adapts/survives within the mixed-species community in response to a dynamically changing environment. This reflects the intricate physiological processes linked to expression of virulence by this bacterium within complex biofilms.     

A dynamic in vitro model for evaluating antimicrobial activity against bacterial biofilms using a new device and clinical-used catheters

The activity of daptomycin compared to vancomycin against Staphylococcus epidermidis-biofilms on intravascular catheters has been evaluated using the new Sevilla device that enables to use medical grade-catheters, in an in vitro model that simulates the in vivo conditions. S. epidermidis-biofilms were obtained on polyurethane catheter segments using the Sevilla device linked to a continuous culture system for 24 h. To assess the antimicrobial activity, at this time the continuous culture system was changed to therapeutic antimicrobial concentration solutions for 48 h. At each 24 h interval time, catheter segments were taken out, washed and sonicated. Viable adherent bacteria were determined by agar plating. Data of surviving bacteria numbers attached to the catheter surface obtained with the Sevilla device showed a very good reproducibility. Daptomycin showed a good activity against S. epidermidis-biofilm on polyurethane catheter surface. After 48 h exposure to daptomycin, surviving adherent bacteria were reduced by 4 log compared to the control with no antimicrobial. Using the same model, vancomycin reduced bacterial survival by only 1.3 log. The Sevilla device enables antimicrobial agent activity against bacterial biofilms grown on the external surface of catheters used in clinical practice to be evaluated. The model used replicates as closely as possible the biofilm formed in a highly standardized way. Using this model, daptomycin demonstrates potent in vitro activity against S. epidermidis-biofilm on a polyurethane catheter; this activity was greater than that showed by vancomycin.