High-Velocity Microsprays Enhance Antimicrobial Activity in Streptococcus mutans Biofilms.

Streptococcus mutans in dental plaque biofilms play a role in caries development. The biofilm's complex structure enhances the resistance to antimicrobial agents by limiting the transport of active agents inside the biofilm. The authors assessed the ability of high-velocity water microsprays to enhance delivery of antimicrobials into 3-d-old S. mutans biofilms. Biofilms were exposed to a 90° or 30° impact, first using a 1-µm tracer bead solution (109 beads/mL) and, second, a 0.2% chlorhexidine (CHX) or 0.085% cetylpyridinium chloride (CPC) solution. For comparison, a 30-s diffusive transport and simulated mouthwash were also performed. Confocal microscopy was used to determine number and relative bead penetration depth into the biofilm. Assessment of antimicrobial penetration was determined by calculating the killing depth detected by live/dead viability staining. The authors first demonstrated that the microspray was able to deliver significantly more microbeads deeper in the biofilm compared with diffusion and mouthwashing exposures. Next, these experiments revealed that the microspray yielded better antimicrobial penetration evidenced by deeper killing inside the biofilm and a wider killing zone around the zone of clearance than diffusion alone. Interestingly the 30° impact in the distal position delivered approximately 16 times more microbeads and yielded approximately 20% more bacteria killing (for both CHX and CPC) than the 90° impact. These data suggest that high-velocity water microsprays can be used as an effective mechanism to deliver microparticles and antimicrobials inside S. mutans biofilms. High shear stresses generated at the biofilm-burst interface might have enhanced bead and antimicrobial delivery inside the remaining biofilm by combining forced advection into the biofilm matrix and physical restructuring of the biofilm itself. Further, the impact angle has potential to be optimized both for biofilm removal and active agents' delivery inside biofilm in those protected areas where some biofilm might remain.

Streptococcus mutans biofilm transient viscoelastic fluid behaviour during high-velocity microsprays.

Using high-speed imaging we assessed Streptococcus mutans biofilm-fluid interactions during exposure to a 60-ms microspray burst with a maximum exit velocity of 51m/s. S. mutans UA159 biofilms were grown for 72h on 10mm-length glass slides pre-conditioned with porcine gastric mucin. Biofilm stiffness was measured by performing uniaxial-compression tests. We developed an in-vitro interproximal model which allowed the parallel insertion of two biofilm-colonized slides separated by a distance of 1mm and enabled high-speed imaging of the removal process at the surface. S. mutans biofilms were exposed to either a water microspray or an air-only microburst. High-speed videos provided further insight into the mechanical behaviour of biofilms as complex liquids and into high-shear fluid-biofilm interaction. We documented biofilms extremely transient fluid behaviour when exposed to the high-velocity microsprays. The presence of time-dependent recoil and residual deformation confirmed the pivotal role of viscoelasticity in biofilm removal. The air-only microburst was effective enough to remove some of the biofilm but created a smaller clearance zone underlying the importance of water and the air-water interface of drops moving over the solid surface in the removal process. Confocal and COMSTAT analysis showed the high-velocity water microspray caused up to a 99.9% reduction in biofilm thickness, biomass and area coverage, within the impact area.

Sequence of oral bacterial co-adhesion and non-contact brushing.

Non-contact plaque removal offers advantages in interproximal spaces, fissures, and pockets. It requires the generation of strong fluid flows and the inclusion of air bubbles to become effective. A pair of co-adhering streptococci and actinomyces has been used previously to demonstrate non-contact removal by sonic brushing. Here we determined the influence of the sequence of co-adhesion of streptococci and actinomyces on non-contact removal from a salivary pellicle by rotary and sonic brushing. After bacterial adhesion, pellicles were brushed in a wet and immersed state, with a distance up to 4 mm to the bristle tips. Bacteria adhering to pellicles from the sequence streptococci followed by actinomyces appeared more difficult to remove and left more large co-aggregates than from the sequence actinomyces followed by streptococci. At contact, rotary and sonic brushing performed equally well in bacterial removal, while at 4 mm, both had lost some efficacy.

Mechanical properties and failure of Streptococcus mutans biofilms, studied using a microindentation device.

Knowledge of mechanical properties and failure mechanisms of biofilms is needed to determine how biofilms react on mechanical stress. Methods currently available cannot be used to determine mechanical properties of biofilms on a small scale with high accuracy. A novel microindentation apparatus in combination with a confocal microscope was used to determine the viscoelastic properties of Streptococcus mutans biofilms. The apparatus comprises a small glass indenter and a highly sensitive force transducer. It was shown that the present biofilm, grown under still conditions, behaves as a viscoelastic solid with a storage modulus of 1-8 kPa and a loss modulus of 5-10 kPa at a strain of 10%. Biofilm failure was investigated visually through a confocal microscope by dragging the indenter through the biofilm. It was shown that the tensile strength of the biofilm is predominantly determined by the tensile strength of the extracellular polysaccharide matrix. The combination of microindentation and confocal microscopy is a promising technique to determine and characterize the mechanical properties of soft materials in various fields of microbiology.

Influence of weight on removal of co-adhering bacteria from salivary pellicles by different modes of brushing.

This study compared removal of pairs of co-adhering and non-co-adhering oral actinomyces and streptococci from salivary pellicles by manual, rotating/oscillating electric and sonic toothbrushes, applying weights up to 240 g. First, actinomyces were allowed to adhere to a pellicle in a parallel plate flow chamber, after which streptococci suspended in saliva were perfused through the chamber at 33 degrees C. On average, 34-39% of the adhering bacteria were adhering as single organisms. For co-adhering and non-co-adhering pairs, 33 and 10% of the adhering bacteria were involved, respectively, in aggregates comprising more than 10 organisms. Brushing by hand removed 82% at low weight (40 g), which was less than by electric (93%) or sonic (92%) brushing, while for all modes of brushing bacterial removal increased with increasing weight to 95-99%. For a non-co-adhering pair, subsequent exposure of brushed pellicles to a streptococcal suspension yielded only 2-16% of bacteria involved in large aggregates, regardless of the mode of brushing. For the co-adhering pair, however, de novo streptococcal adhesion to hand-brushed pellicles yielded 34-57% of bacteria involved in large aggregates, while electric and sonic brushing left 22-35% of the bacteria involved in large aggregates. De novo streptococcal adhesion for the co-adhering pair increased with increasing weight for the electric and sonic brush in contrast to the manual brush. Since a strong influence of co-adhesion is evident in de novo streptococcal adhesion, despite nearly complete removal of all actinomyces, these observations suggest that the three modes of brushing leave footprints to which streptococci preferentially adhere.

Non-contact removal of coadhering and non-coadhering bacterial pairs from pellicle surfaces by sonic brushing and de novo adhesion.

Coadhesion between oral microbial pairs is an established factor in the spatiotemporal development and prevalence of mixed-species communities in early dental plaque in vivo. This study compares removal and de novo adhesion of pairs of coadhering and non-coadhering oral actinomyces and streptococci by sonic brushing on salivary pellicles in a non-contact mode as a function of the distance between the brush and the pellicle surface in vitro. First, actinomycetes were adhered to a pellicle surface, after which streptococci suspended in saliva were allowed to adhere. Removal was examined by non-contact, sonic brushing with a wetted brush on a either a wetted or a substratum immersed to a depth of 7 mm. After brushing, de novo adhesion of streptococci to brushed pellicles was studied. For coadhering and non-coadhering pairs, 34% and 9%, respectively, of the adhering bacteria were involved in aggregates comprising more than 10 organisms. Non-contact, sonic brushing removed up to 99% of the adhering bacteria, regardless of the state of immersion of the substratum. Bacterial removal decreased with increasing distance of up to 6 mm between brush and pellicle surface. For the non-coadhering pair, subsequent exposure of pellicles to a streptococcal suspension yielded about 6% of bacteria involved in large aggregates. Alternatively, de novo adhesion of the coadhering streptococcal strain to pellicles brushed on the wetted substratum yielded 31% of bacteria involved in large aggregates, but after brushing the immersed substratum only 12% of the adhering bacteria were found in large aggregates. It is concluded that non-contact sonic brushing, under immersion, removes high percentage of adhering bacterial pairs up to a distance of 6 mm between the brush and the pellicle surface. However, non-contact, sonic brushing with only a thin wet film on the substratum may leave footprints to which streptococci preferentially adhere.

Pathogenesis and prevention of biomaterial centered infections.

One of the major drawbacks in the use of biomedical materials is the occurrence of biomaterials centered infections. After implantation, the host interacts with a biomaterial by forming a conditioning film on its surface and an immune reaction towards the foreign material. When microorganisms can reach the biomaterials surface they can adhere to it. Adhesion of microorganisms to an implant is mediated by their physico-chemical surface properties and the properties of the biomaterials surface itself. Subsequent surface growth of the microorganisms will lead to a mature biofilm and infection, which is difficult to eradicate by antibiotics. The purpose of this review is to give an overview of the mechanisms involved in biomaterials centered infection and the possible methods to prevent these infections.

Late hematogenous infection of subcutaneous implants in rats.

Late biomaterial-centered infection is a major complication associated with the use of biomaterial implants. In this study biomaterials that had been implanted subcutaneously in rats were hematogenously challenged with bacteria 4 weeks after implantation. Bacteria were spread either by intravenous injection or by stimulation of bacterial translocation. It was found that none of the biomaterials was infected by hematogenous spread, whereas 5% of the implants were infected by perioperative contamination. We conclude that late hematogenous infection of subcutaneous biomaterials does not occur in the rat. For humans as well, there are growing doubts whether implants actually become infected through hematogenous routes; it is thought that late infections may be caused by delayed appearance of perioperatively introduced bacteria.

Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria.

The infection of biomaterials is determined by an interplay of adhesion and surface growth of the infecting organisms. In this study, the antimicrobial effects on adhering bacteria of a positively charged poly(methacrylate) surface (xi potential +12 mV) were compared with those of negatively charged poly(methyl methacrylate) (-12 mV) and a highly negatively charged poly(methacrylate) (-18 mV) surface. Initial adhesion of Staphylococcus aureus ATCC 12600, Staphylococcus epidermidis HBH(2) 102, Escherichia coli O2K2 and Pseudomonas aeruginosa AK1 to these surfaces was measured in a parallel plate flow chamber in phosphate-buffered saline. Adhering bacteria were allowed to multiply by perfusing the flow chamber with growth medium. All bacteria adhered most rapidly to the positively charged surface, but there was no subsequent surface growth of the Gram-negative strains. On the negatively charged surfaces, despite a slower initial adhesion, surface growth of the adhering bacteria was exponential for both Gram-positive and Gram-negative strains. These results suggest that positively charged biomaterial surfaces exert an antimicrobial effect on adhering Gram-negative bacteria, but not on Gram-positive ones.

Pooled human immunoglobulins reduce adhesion of Pseudomonas aeruginosa in a parallel plate flow chamber.

The influence of pooled polyclonal immunoglobulin (IgG) interactions with both bacteria and model substrates in altering Pseudomonas aeruginosa surface adhesion is reported. Opsonization of this pathogen by polyclonal human IgG and preadsorption of IgG to glass surfaces both effectively reduce initial deposition rates and surface growth of P. aeruginosa IFO3455 from dilute nutrient broth in a parallel plate flow chamber. Polyclonal IgG depleted of P. aeruginosa-specific antibodies reduces the initial deposition rate or surface growth to levels intermediate between exposed and nonexposed IgG conditions. Bacterial surface properties are changed in the presence of opsonizing IgG. Plateau contact angle analysis via sessile drop technique shows a drop in P. aeruginosa surface hydrophobicity after IgG exposure consistent with a more hydrophilic IgG surface coat. Zeta potential values for opsonized versus nonopsonized bacteria exhibit little change. X-ray photoelectron spectroscopy measurements provide surface compositional evidence for IgG attachment to bacterial surfaces. Surface elemental ratios attributed to IgG protein signals versus those attributed primarily to bacterial polysaccharide surface or lipid membrane change with IgG opsonization. Direct evidence for antibody-modified P. aeruginosa surface properties correlates both with reduction of bacterial adhesion to glass surfaces under flow in nutrient medium reported and previous reports of IgG efficacy against P. aeruginosa motility in vitro and infection in vivo.

Initial adhesion and surface growth of Staphylococcus epidermidis and Pseudomonas aeruginosa on biomedical polymers.

The infection risk of biomaterials implants varies between different materials and is determined by an interplay of adhesion and surface growth of the infecting organisms. In this study, we compared initial adhesion and surface growth of Staphylococcus epidermidis HBH(2) 102 and Pseudomonas aeruginosa AK1 on poly(dimethylsiloxane), Teflon, polyethylene, polypropylene, polyurethane, poly(ethylene terephthalate), poly(methyl methacrylate), and glass. Initial adhesion was measured in situ in a parallel plate flow chamber with microorganisms suspended in phosphate-buffered saline, while subsequent surface growth was followed in full and in 20 times diluted growth medium. Initial adhesion of both bacterial strains was similar to all biomaterials. In full growth medium, generation times of surface growing S. epidermidis ranged from 17 to 38 min with no relation to wettability, while in diluted growth medium generation times increased from 44 to 98 min with increasing surface wettability. For P. aeruginosa no influence of surface wettability on generation times was observed, but generation times increased with decreasing desorption rates, maximal generation times being 47 min and minimal values down to 30 min. Generally, generation times of adhering bacteria were shorter than of planktonic bacteria. In conclusion, surface growth of initially adhering bacteria is influenced by biomaterials surface properties to a greater extent than initial adhesion.

Initial adhesion and surface growth of Pseudomonas aeruginosa on negatively and positively charged poly(methacrylates).

The infection risk of biomaterial implants is determined by an interplay of bacterial adhesion and surface growth of the adhering organisms. In this study, we compared initial adhesion and surface growth of Pseudomonas aeruginosa AK1 (zeta potential -7 mV) on negatively charged (PMMA/MAA, zeta potential -18 mV) and positively charged (PMMA/TMAEMA-Cl, zeta-potential +12 mV) methacrylate copolymers in situ in a parallel plate flow chamber. Initial adhesion was measured using phosphate-buffered saline and subsequent surface growth of the adhering bacteria using nutrient broth as growth medium. Initial adhesion was twice as fast on the positively charged methacrylate than on the negatively charged copolymer. Surface growth, however, was absent on the positively charged copolymer, while on the negatively charged methacrylate the number of bacteria increased exponentially during surface growth with a generation time of 32 min. From the results of this study it can be concluded that positively charged biomaterial surfaces might show reduced risks of biomaterials-centred infections, despite being more adhesive.