An in vitro biofilm model system to facilitate study of microbial communities of the human oral cavity.

The human oral cavity is host to a diverse microbiota. Much of what is known about the behaviour of oral microbes derives from studies of individual or several cultivated species, situations which do not totally reflect the function of organisms within more complex microbiota or multispecies biofilms. The number of validated models that allow examination of the role that biofilms play during oral cavity colonization is also limited. The CDC biofilm reactor is a standard method that has been deployed to study interactions between members of human microbiotas allowing studies to be completed during an extended period under conditions where nutrient availability, and washout of waste products are controlled. The objective of this work was to develop a robust in vitro biofilm-model system from a pooled saliva inoculum to study the development, reproducibility and stability of the oral microbiota. By employing deep sequencing of the variable regions of the 16S rRNA gene, we found that the CDC biofilm reactor could be used to efficiently cultivate microbiota containing all six major phyla previously identified as the core saliva microbiota. After an acclimatisation period, communities in each reactor stabilised. Replicate reactors were predominately populated by a shared core microbiota; variation between replicate reactors was primarily driven by shifts in abundance of shared operational taxonomic units. We conclude that the CDC biofilm reactor can be used to cultivate communities that replicate key features of the human oral cavity and is a useful tool to facilitate studies of the dynamics of these communities.

What can an echocardiographer see in briefly presented stimuli? Perceptual expertise in dynamic search.

BACKGROUND: Experts in medical image perception are able to detect abnormalities rapidly from medical images. This ability is likely due to enhanced pattern recognition on a global scale. However, the bulk of research in this domain has focused on static rather than dynamic images, so it remains unclear what level of information that can be extracted from these displays. This study was designed to examine the visual capabilities of echocardiographers-practitioners who provide information regarding cardiac integrity and functionality. In three experiments, echocardiographers and naïve participants completed an abnormality detection task that comprised movies presented on a range of durations, where half were abnormal. This was followed by an abnormality categorization task. RESULTS: Across all durations, the results showed that performance was high for detection, but less so for categorization, indicating that categorization was a more challenging task. Not surprisingly, echocardiographers outperformed naïve participants. CONCLUSIONS: Together, this suggests that echocardiographers have a finely tuned capability for cardiac dysfunction, and a great deal of visual information can be extracted during a global assessment, within a brief glance. No relationship was evident between experience and performance which suggests that other factors such as individual differences need to be considered for future studies.

The efficacy of topical agents used in wounds for managing chronic biofilm infections: A systematic review.

OBJECTIVES: Clinicians have increasingly adopted the widespread use of topical agents to manage chronic wound infections, despite limited data on their effectiveness in vivo. This study sought to evaluate the evidence for commonly employed topical agents used in wounds for the purpose of treating chronic infections caused by biofilm. METHOD: We included in vitro, animal and human in vivo studies where topical agents were tested for their efficacy against biofilms, for use in wound care. For human studies, we only included those which utilised appropriate identification techniques for visualising and confirming the presence of biofilms. RESULT: A total of 640 articles were identified, with 43 included after meeting eligibility. In vitro testing accounted for 90% (n = 39) of all included studies, five studies using animal models and three human in vivo studies. Sixteen different laboratory models were utilised, with the most frequent being the minimum biofilm eradication concentration (MBEC™) / well plate assay (38%, n = 15 of 39). A total of 44 commercially available topical agents were grouped into twelve categories with the most commonly tested agents being silver, iodine and polyhexamethylene biguanide (PHMB). In vitro results on efficacy demonstrated iodine as having the highest mean log10 reductions of all agents (4.81, ±3.14). CONCLUSION: There is large disparity in the translation of laboratory studies to researchers undertaking human trials relating to the effectiveness of commercially available topical agents. There is insufficient human in vivo evidence to definitively recommend any commercially available topical agent over another for the treatment of chronic wound biofilms. The heterogeneity identified between study designs (in vitro to in vivo) further limits the generalisability of results.

Mathematical modeling of dispersal phenomenon in biofilms.

A mathematical model for dispersal phenomenon in multispecies biofilm based on a continuum approach and mass conservation principles is presented. The formation of dispersed cells is modeled by considering a mass balance for the bulk liquid and the biofilm. Diffusion of these cells within the biofilm and in the bulk liquid is described using a diffusion-reaction equation. Diffusion supposes a random character of mobility. Notably, biofilm growth is modeled by a hyperbolic partial differential equation while the diffusion process of dispersed cells by a parabolic partial differential equation. The two are mutually connected but governed by different equations that are coupled by two growth rate terms. Three biological processes are discussed. The first is related to experimental observations on starvation induced dispersal [1]. The second considers diffusion of a non-lethal antibiofilm agent which induces dispersal of free cells. The third example considers dispersal induced by a self-produced biocide agent.

The prevalence of biofilms in chronic wounds: a systematic review and meta-analysis of published data.

The presence of biofilms in chronic non-healing wounds, has been identified through in vitro model and in vivo animal data. However, human chronic wound studies are under-represented and generally report low sample sizes. For this reason we sought to ascertain the prevalence of biofilms in human chronic wounds by undertaking a systematic review and meta-analysis. Our initial search identified 554 studies from the literature databases (Cochrane Library, Embase, Medline). After removal of duplicates, and those not meeting the requirements of inclusion, nine studies involving 185 chronic wounds met the inclusion criteria. Prevalence of biofilms in chronic wounds was 78.2 % (confidence interval [CI 61.6-89, p<0.002]). The results of our meta-analysis support our clinical assumptions that biofilms are ubiquitous in human chronic non-healing wounds.

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.

Cold water cleaning of brain proteins, biofilm and bone - harnessing an ultrasonically activated stream.

In the absence of sufficient cleaning of medical instruments, contamination and infection can result in serious consequences for the health sector and remains a significant unmet challenge. In this paper we describe a novel cleaning system reliant on cavitation action created in a free flowing fluid stream where ultrasonic transmission to a surface, through the stream, is achieved using careful design and control of the device architecture, sound field and the materials employed. Cleaning was achieved with purified water at room temperature, moderate fluid flow rates and without the need for chemical additives or the high power consumption associated with conventional strategies. This study illustrates the potential in harnessing an ultrasonically activated stream to remove biological contamination including brain tissue from surgical stainless steel substrates, S. epidermidis biofilms from glass, and fat/soft tissue matter from bone structures with considerable basic and clinical applications.

Removal of Dental Biofilms with an Ultrasonically Activated Water Stream.

Acidogenic bacteria within dental plaque biofilms are the causative agents of caries. Consequently, maintenance of a healthy oral environment with efficient biofilm removal strategies is important to limit caries, as well as halt progression to gingivitis and periodontitis. Recently, a novel cleaning device has been described using an ultrasonically activated stream (UAS) to generate a cavitation cloud of bubbles in a freely flowing water stream that has demonstrated the capacity to be effective at biofilm removal. In this study, UAS was evaluated for its ability to remove biofilms of the cariogenic pathogen Streptococcus mutans UA159, as well as Actinomyces naeslundii ATCC 12104 and Streptococcus oralis ATCC 9811, grown on machine-etched glass slides to generate a reproducible complex surface and artificial teeth from a typodont training model. Biofilm removal was assessed both visually and microscopically using high-speed videography, confocal scanning laser microscopy (CSLM), and scanning electron microscopy (SEM). Analysis by CSLM demonstrated a statistically significant 99.9% removal of S. mutans biofilms exposed to the UAS for 10 s, relative to both untreated control biofilms and biofilms exposed to the water stream alone without ultrasonic activation (P < 0.05). The water stream alone showed no statistically significant difference in removal compared with the untreated control (P = 0.24). High-speed videography demonstrated a rapid rate (151 mm(2) in 1 s) of biofilm removal. The UAS was also highly effective at S. mutans, A. naeslundii, and S. oralis biofilm removal from machine-etched glass and S. mutans from typodont surfaces with complex topography. Consequently, UAS technology represents a potentially effective method for biofilm removal and improved oral hygiene.

An experimental and computational study of the hydrodynamics of high-velocity water microdrops for interproximal tooth cleaning.

The flow field and local hydrodynamics of high-velocity water microdrops impacting the interproximal (IP) space of typodont teeth were studied experimentally and computationally. Fourteen-day old Streptococcus mutans biofilms in the IP space were treated by a prototype AirFloss delivering 115 µL of water at a maximum exit-velocity of 60 ms(-1) in a 33-ms burst. Using high-speed imaging, footage was generated showing the details of the burst, and demonstrating the removal mechanism of the biofilms. Footage was also generated to characterize the viscoelastic behavior of the biofilms when impacted by an air-only burst, which was compared to the water burst. Image analysis demonstrated the importance of fluid forces on the removal pattern of interdental biofilms. X-ray micro-Computed Tomography (µ-CT) was used to obtain 3D images of the typodont and the IP spaces. Computational Fluid Dynamics (CFD) simulations were performed to study the effect of changing the nozzle position and design on the hydrodynamics within the IP space. Results confirmed our previous data regarding the wall shear stress generated by high-velocity water drops which dictated the efficacy of biofilm detachment. Finally, we showed how CFD models could be used to optimize water drop or burst design towards a more effective biofilm removal performance.

Antibiotic-loaded synthetic calcium sulfate beads for prevention of bacterial colonization and biofilm formation in periprosthetic infections.

Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 10(6) CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms; therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection.

Surface-attached cells, biofilms and biocide susceptibility: implications for hospital cleaning and disinfection.

Microbes tend to attach to available surfaces and readily form biofilms, which is problematic in healthcare settings. Biofilms are traditionally associated with wet or damp surfaces such as indwelling medical devices and tubing on medical equipment. However, microbes can survive for extended periods in a desiccated state on dry hospital surfaces, and biofilms have recently been discovered on dry hospital surfaces. Microbes attached to surfaces and in biofilms are less susceptible to biocides, antibiotics and physical stress. Thus, surface attachment and/or biofilm formation may explain how vegetative bacteria can survive on surfaces for weeks to months (or more), interfere with attempts to recover microbes through environmental sampling, and provide a mixed bacterial population for the horizontal transfer of resistance genes. The capacity of existing detergent formulations and disinfectants to disrupt biofilms may have an important and previously unrecognized role in determining their effectiveness in the field, which should be reflected in testing standards. There is a need for further research to elucidate the nature and physiology of microbes on dry hospital surfaces, specifically the prevalence and composition of biofilms. This will inform new approaches to hospital cleaning and disinfection, including novel surfaces that reduce microbial attachment and improve microbial detachment, and methods to augment the activity of biocides against surface-attached microbes such as bacteriophages and antimicrobial peptides. Future strategies to address environmental contamination on hospital surfaces should consider the presence of microbes attached to surfaces, including biofilms.

Removal of interproximal dental biofilms by high-velocity water microdrops.

The influence of the impact of a high-velocity water microdrop on the detachment of Streptococcus mutans UA159 biofilms from the interproximal (IP) space of teeth in a training typodont was studied experimentally and computationally. Twelve-day-old S. mutans biofilms in the IP space were exposed to a prototype AirFloss delivering 115 µL water at a maximum exit velocity of 60 m/sec in a 30-msec burst. Using confocal microscopy and image analysis, we obtained quantitative measurements of the percentage removal of biofilms from different locations in the IP space. The 3D geometry of the typodont and the IP spaces was obtained by micro-computed tomography (µ-CT) imaging. We performed computational fluid dynamics (CFD) simulations to calculate the wall shear stress (τw ) distribution caused by the drops on the tooth surface. A qualitative agreement and a quantitative relationship between experiments and simulations were achieved. The wall shear stress (τw ) generated by the prototype AirFloss and its spatial distribution on the teeth surface played a key role in dictating the efficacy of biofilm removal in the IP space.

On the mechanics of bacterial biofilms on non-dissolvable surgical sutures: a laser scanning confocal microscopy-based finite element study.

Biofilms are bacterial communities encapsulated within a self-secreted extracellular polymeric substance and are responsible for a wide range of chronic medical device related infections. Understanding and addressing the conditions that lead to the attachment and detachment of biofilms from biomedical surfaces (orthopaedic implants, sutures, intravenous catheters, cardio-vascular stents) has the potential to identify areas of the device that might be more prone to infection and predict how and when biofilms might dislodge. In this study, an integrated software methodology was devised to create image-based microscopic finite element models of real biofilm colonies of Staphylococcus aureus attached to a fragment of surgical suture. The goal was to predict how deformation of the suture may lead to the potential detachment of biofilm colonies by solving the equations of continuum mechanics using the finite element method for various loading cases. Tension, torsion and bending of the biomaterial structure were simulated, demonstrating that small strains in the suture can produce surface shear stresses sufficient to trigger the sliding of biofilms over the suture surface. Applications of this technique to other medical devices are discussed.

Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

Clinical implications of power toothbrushing on fluoride delivery: effects on biofilm plaque metabolism and physiology.

Dental biofilms are implicated in the formation of caries and periodontal disease. A major constituent of the supragingival biofilm is Streptococcus mutans, which produces lactic acid from sucrose fermentation, enhancing enamel demineralization and eventual caries development. Caries prevention through F inhibits enamel demineralization and promotes remineralization. Fluoride also exerts effects on metabolic activities in the supragingival biofilm such as aerobic respiration, acid fermentation and dentrification. In experimental S. mutans biofilms, adding 1000 ppm F to an acidogenic biofilm resulting from 10% sucrose addition increased pH to pre-sucrose levels, suggesting inhibition of acid fermentation. F effects on metabolic activity and sucrose utilization in interproximal plaque biofilms were also recorded. Addition of 10% sucrose reduced pH from neutral to 4.2, but subsequent addition of 1000 ppm F increased pH by 1 unit, inhibiting acid fermentation. 10% Sucrose addition also stimulated denitrification, increasing production of nitrous oxide (N(2)O). Addition of 1000 ppm F suppressed denitrification, indicating an additional mechanism by which F exerts effects in the active interproximal biofilm. Finally, fluid dynamic activity by power tooth brushing enhanced F delivery and retention in an experimental S. mutans biofilm, suggesting a potential novel benefit for this intervention beyond mechanical plaque removal.

The high-affinity phosphate transporter Pst in Proteus mirabilis HI4320 and its importance in biofilm formation.

Proteus mirabilis causes urinary tract infections (UTIs) in individuals requiring long-term indwelling catheterization. The pathogenesis of this uropathogen is mediated by a number of virulence factors and the formation of crystalline biofilms. In addition, micro-organisms have evolved complex systems for the acquisition of nutrients, including the phosphate-specific transport system, which has been shown to be important in biofilm formation and pathogenesis. A functional Pst system is important during UTIs caused by P. mirabilis HI4320, since transposon mutants in the PstS periplasmic binding protein and the PstA permease protein were attenuated in the CBA mouse model of UTI. These mutants displayed a defect in biofilm formation when grown in human urine. This study focuses on a comparison of the proteomes during biofilm and planktonic growth in phosphate-rich medium and human urine, and microscopic investigations of biofilms formed by the pst mutants. Our data suggest that (i) the Deltapst mutants, and particularly the DeltapstS mutant, are defective in biofilm formation, and (ii) the proteomes of these mutants differ significantly from that of the wild-type. Therefore, since the Pst system of P. mirabilis HI4320 negatively regulates biofilm formation, this system is important for the pathogenesis of these organisms during complicated UTIs.

Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro.

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as 'negative for biofilm formation' based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.

Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress.

A digital image correlation (DIC) method was applied to characterize the mechanical behavior of Pseudomonas aeruginosa biofilms in response to wall shear stress using digital video micrographs taken from biofilm flow cells. The appearance of the biofilm in the transmitted light photomicrographs presented a natural texture which was highly conducive to random encoding for DIC. The displacement fields were calculated for two biofilm specimens. The DIC method concurred with previous analysis showing that biofilms exhibit viscoelastic behavior, but had the advantage over simple length measurements of longitudinal strain that it could precisely measure local strains in length (x) and width (y) within biofilm clusters with a 2 mum resolution as a function of time and wall shear stress. It was concluded that DIC was more accurate at measuring elastic moduli than simple length measurements, but that time-lapse 3D images would enable even more accurate estimates to be performed.