Microbial adhesion to surface-grafted polyacrylamide brushes after long-term exposure to PBS and reconstituted freeze-dried saliva

Polyacrylamide (PAAm) brushes, covalently grafted from silicon wafer surfaces were examined for their ability to inhibit microbial adhesion after long-term exposure to PBS or reconstituted freeze-dried saliva for time intervals from 48 h up to 1 month at 37 degrees C. Microbial adhesion after exposure was studied in a parallel plate flow chamber. Infrared spectra showed that PAAm brushes exhibit good chemical stability upon incubation in both PBS and reconstituted freeze-dried saliva up to 1 month. Reductions in microbial adhesion on PAAm brushes after exposure to PBS or reconstituted freeze-dried saliva varied from 63 to 93% depending on the microbial strain considered, even after 1 month of exposure of the brushes to reconstituted freeze-dried saliva. (C) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 94A: 997-1000,2010

Streptococcus mutans adhesion force sensing in multi-species oral biofilms

Bacteria utilize chemical and mechanical mechanisms to sense their environment, to survive hostile conditions. In mechanical sensing, intra-bilayer pressure profiles change due to deformation induced by the adhesion forces bacteria experience on a surface. Emergent properties in mono-species Streptococcus mutans biofilms, such as extracellular matrix production, depend on the adhesion forces that streptococci sense. Here we determined whether and how salivary-conditioning film (SCF) adsorption and the multi-species nature of oral biofilm influence adhesion force sensing and associated gene expression by S. mutans. Hereto, Streptococcus oralis, Actinomyces naeslundii, and S. mutans were grown together on different surfaces in the absence and presence of an adsorbed SCF. Atomic force microscopy and RT-qPCR were used to measure S. mutans adhesion forces and gene expressions. Upon SCF adsorption, stationary adhesion forces decreased on a hydrophobic and increased on a hydrophilic surface to around 8 nN. Optical coherence tomography showed that triple-species biofilms on SCF-coated surfaces with dead S. oralis adhered weakly and often detached as a contiguous sheet. Concurrently, S. mutans displayed no differential adhesion force sensing on SCF-coated surfaces in the triple-species biofilms with dead S. oralis, but once live S. oralis were present S. mutans adhesion force sensing and gene expression ranked similar as on surfaces in the absence of an adsorbed SCF. Concluding, live S. oralis may enzymatically degrade SCF components to facilitate direct contact of biofilm inhabitants with surfaces and allow S. mutans adhesion force sensing of underlying surfaces to define its appropriate adaptive response. This represents a new function of initial colonizers in multi-species oral biofilms

Treating natural disaster victims is dealing with shortages:An orthopaedics perspective

During natural disasters such as earthquakes or tsunamis, most of the casualties are known to suffer from musculoskeletal injuries. This leads to an enormous need of orthopaedic (surgical) implants such as osteosynthesis plates, which are difficult to provide in developing countries that rely on imported ones. One of the alternatives is utilization of local resources, but only after they have been proven safe to use, and meet the international standards set. Through this paper we would like to urge the international community to include locally produced biomedical products, like osteosynthesis plates in their scientific evaluations and communications. When the quality of local products is proven, the reluctance to use local products also by surgeons from developing countries will disappear and larger scale production can be initiated. This in its turn solves many problems that come after natural disasters and stimulates the national economy in an efficient and effective way

The role of small-colony variants in failure to diagnose and treat biofilm infections in orthopedics

Biomaterial-related infection of joint replacements is the second most common cause of implant failure, with serious consequences. Chronically infected replacements cannot be treated without removal of the implant, as the bio film mode of growth protects the bacteria against antibiotics. This review discusses bio film formation on joint replacements and the important clinical phenomenon of small-colony variants (SCVs). These slow-growing phenotypic variants often remain undetected or are misdiagnosed using hospital microbiological analyses due to their unusual morphological appearance and biochemical reactions. In addition, SCVs make the infection difficult to eradicate. They often lead to recurrence since they respond poorly to standard antibiotic treatment and can sometimes survive intracellularly

X-Ray Photoelectron Spectroscopy on Microbial Cell Surfaces:A Forgotten Method for the Characterization of Microorganisms Encapsulated With Surface-Engineered Shells

Encapsulation of single microbial cells by surface-engineered shells has great potential for the protection of yeasts and bacteria against harsh environmental conditions, such as elevated temperatures, UV light, extreme pH values, and antimicrobials. Encapsulation with functionalized shells can also alter the surface characteristics of cells in a way that can make them more suitable to perform their function in complex environments, including bio-reactors, bio-fuel production, biosensors, and the human body. Surface-engineered shells bear as an advantage above genetically-engineered microorganisms that the protection and functionalization added are temporary and disappear upon microbial growth, ultimately breaking a shell. Therewith, the danger of creating a "super-bug," resistant to all known antimicrobial measures does not exist for surface-engineered shells. Encapsulating shells around single microorganisms are predominantly characterized by electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, particulate micro-electrophoresis, nitrogen adsorption-desorption isotherms, and X-ray diffraction. It is amazing that X-ray Photoelectron Spectroscopy (XPS) is forgotten as a method to characterize encapsulated yeasts and bacteria. XPS was introduced several decades ago to characterize the elemental composition of microbial cell surfaces. Microbial sample preparation requires freeze-drying which leaves microorganisms intact. Freeze-dried microorganisms form a powder that can be easily pressed in small cups, suitable for insertion in the high vacuum of an XPS machine and obtaining high resolution spectra. Typically, XPS measures carbon, nitrogen, oxygen and phosphorus as the most common elements in microbial cell surfaces. Models exist to transform these compositions into well-known, biochemical cell surface components, including proteins, polysaccharides, chitin, glucan, teichoic acid, peptidoglycan, and hydrocarbon like components. Moreover, elemental surface compositions of many different microbial strains and species in freeze-dried conditions, related with zeta potentials of microbial cells, measured in a hydrated state. Relationships between elemental surface compositions measured using XPS in vacuum with characteristics measured in a hydrated state have been taken as a validation of microbial cell surface XPS. Despite the merits of microbial cell surface XPS, XPS has seldom been applied to characterize the many different types of surface-engineered shells around yeasts and bacteria currently described in the literature. In this review, we aim to advocate the use of XPS as a forgotten method for microbial cell surface characterization, for use on surface-engineered shells encapsulating microorganisms

Two-Stage Interpretation of Changes in TEER of Intestinal Epithelial Layers Protected by Adhering Bifidobacteria During E. coli Challenges

Mechanisms of gastrointestinal protection by probiotic bacteria against infection involve amongst others, modulation of intestinal epithelial barrier function. Trans-epithelial electrical resistance (TEER) is widely used to evaluate cellular barrier functions. Here, we developed a two-stage interpretative model of the time-dependence of the TEER of epithelial layers grown in a transwell during Escherichia coli challenges in the absence or presence of adhering bifidobacteria. E. coli adhesion in absence or presence of adhering bifidobacteria was enumerated using selective plating. After 4-8 h, E. coli challenges increased TEER to a maximum due to bacterial adhesion and increased expression of a tight-junction protein [zonula occludens-1 (ZO-1)], concurrent with a less dense layer structure, that is indicative of mild epithelial layer damage. Before the occurrence of a TEER-maximum, decreases in electrical conductance (i.e., the reciprocal TEER) did not relate with para-cellular dextran-permeability, but after occurrence of a TEER-maximum, dextran-permeability and conductance increased linearly, indicative of more severe epithelial layer damage. Within 24 h after the occurrence of a TEER maximum, TEER decreased to below the level of unchallenged epithelial layers demonstrating microscopically observable holes and apoptosis. Under probiotic protection by adhering bifidobacteria, TEER-maxima were delayed or decreased in magnitude due to later transition from mild to severe damage, but similar linear relations between conductance and dextran permeability were observed as in absence of adhering bifidobacteria. Based on the time-dependence of the TEER and the relation between conductance and dextran-permeability, it is proposed that bacterial adhesion to epithelial layers first causes mild damage, followed by more severe damage after the occurrence of a TEER-maximum. The mild damage caused by E. coli prior to the occurrence of TEER maxima was reversible upon antibiotic treatment, but the severe damage after occurrence of TEER maxima could not be reverted by antibiotic treatment. Thus, single-time TEER is interpretable in two ways, depending whether increasing to or decreasing from its maximum. Adhering bifidobacteria elongate the time-window available for antibiotic treatment to repair initial pathogen damage to intestinal epithelial layers.</p

Role of adhesion forces in mechanosensitive channel gating inStaphylococcus aureusadhering to surfaces

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials

A quantitative model for the surface restructuring of repeatedly plasma treated silicone rubber

Surface restructuring in ambient air of medical grade silicone rubber surfaces modified by repeated RF plasma treatments using various discharge gases including oxygen, argon, carbon dioxide and ammonia, was studied quantitatively. From advancing and receding water contact angle data, the fraction of the surface covered by mobile and immobile polar groups, and a characteristic time constant of the restructuring process were calculated. For argon plasma treated surfaces, the fraction of immobile polar groups increased with repeated plasma treatments, but remained relatively constant for samples repeatedly treated by an ammonia plasma. The use of an oxygen plasma only yielded incorporation of mobile polar groups but not of immobile polar groups. The increase in the restructuring time constants of argon and ammonia plasma treated silicone rubber with the number of plasma treatments suggested enhanced crosslinking of the silicone rubber by these plasmas. In contrast, when an oxygen plasma was repeatedly used, the restructuring time constant decreased suggesting chain cleavage by an oxygen plasma. Tentatively, the carbon dioxide plasma treatment of silicone rubber may initially (up to 3-4 repeated treatments) yield chain cleavage, while the occurrence of crosslinking is indicated after more repetitions.</p

Emergent Properties in Streptococcus mutans Biofilms Are Controlled through Adhesion Force Sensing by Initial Colonizers

Bacterial adhesion is accompanied by altered gene expression, leading to "emergent" properties of biofilm bacteria that are alien to planktonic ones. With the aim of revealing the role of environmental adhesion forces in emergent biofilm properties, genes in Streptococcus mutans UA159 and a quorum-sensing-deficient mutant were identified that become expressed after adhesion to substratum surfaces. Using atomic force microscopy, adhesion forces of initial S. mutans colonizers on four different substrata were determined and related to gene expression. Adhesion forces upon initial contact were similarly low across different substrata, ranging between 0.2 and 1.2 nN regardless of the strain considered. Bond maturation required up to 21 s, depending on the strain and substratum surface involved, but stationary adhesion forces also were similar in the parent and in the mutant strain. However, stationary adhesion forces were largest on hydrophobic silicone rubber (19 to 20 nN), while being smallest on hydrophilic glass (3 to 4 nN). brpA gene expression in thin (34 to 48 mu m) 5-h S. mutans UA159 biofilms was most sensitive to adhesion forces, while expression of gbpB and comDE expressions was weakly sensitive. ftf, gtfB, vicR, and relA expression was insensitive to adhesion forces. In thicker (98 to 151 mu m) 24-h biofilms, adhesion-force-induced gene expression and emergent extracellular polymeric substance (EPS) production were limited to the first 20 to 30 mu m above a substratum surface. In the quorum-sensing-deficient S. mutans, adhesionforce-controlled gene expression was absent in both 5- and 24-h biofilms. Thus, initial colonizers of substratum surfaces sense adhesion forces that externally trigger emergent biofilm properties over a limited distance above a substratum surface through quorum sensing. IMPORTANCE A new concept in biofilm science is introduced: "adhesion force sensitivity of genes," defining the degree up to which expression of different genes in adhering bacteria is controlled by the environmental adhesion forces they experience. Analysis of gene expression as a function of height in a biofilm showed that the information about the substratum surface to which initially adhering bacteria adhere is passed up to a biofilm height of 20 to 30 mu m above a substratum surface, highlighting the importance and limitations of cell-to-cell communication in a biofilm. Bacteria in a biofilm mode of growth, as opposed to planktonic growth, are responsible for the great majority of human infections, predicted to become the number one cause of death in 2050. The concept of adhesion force sensitivity of genes provides better understanding of bacterial adaptation in biofilms, direly needed for the design of improved therapeutic measures that evade the recalcitrance of biofilm bacteria to antimicrobials