Evaluation of a nonthermal plasma needle to eliminate ex vivo biofilms in root canals of extracted human teeth.

AIM: To evaluate the efficacy of a nonthermal plasma (NTP) at atmospheric pressure on ex vivo biofilm in root canals of extracted teeth. METHODOLOGY: Intracanal contents from three teeth with root canal infections were collected, pooled and grown in thirty-five microCT-mapped root canals of extracted and instrumented human teeth. One group of teeth was treated with NTP, another with 6% NaOCl and one set was left untreated. The intracanal contents from twenty-seven teeth (nine teeth in each group) were plated on agar and colony forming units were determined. Parametric test of one-way analysis of variance (anova) was used to analyse statistical significance. The remaining teeth were cut open, stained with LIVE/DEAD(®) and examined with confocal laser scanning microscopy. RESULTS: The untreated root canals were covered with biofilm of varying thickness. Treatment with nonthermal plasma decreased the number of viable bacteria in biofilms by one order of magnitude, whilst the NaOCl control achieved a reduction of more than four magnitudes. Both the NTP and the NaOCl treatment results were significantly different from the negative control (P < 0.05). CONCLUSION: The nonthermal plasma displayed antimicrobial activity against endodontic biofilms in root canals, but was not as effective as the use of 6% NaOCl.

The polymicrobial nature of biofilm infection.

The model of biofilm infection was first proposed over a decade ago. Recent scientific advances have added much to our understanding of biofilms, usually polymicrobial communities, which are commonly associated with chronic infection. Metagenomics has demonstrated that bacteria pursuing a biofilm strategy possess many mechanisms for encouraging diversity. By including multiple bacterial and/or fungal species in a single community, biofilms obtain numerous advantages, such as passive resistance, metabolic cooperation, byproduct influence, quorum sensing systems, an enlarged gene pool with more efficient DNA sharing, and many other synergies, which give them a competitive advantage. Routine clinical cultures are ill-suited for evaluating polymicrobial infections. DNA methods utilizing PCR methods, PCR/mass spectroscopy and sequencing have demonstrated their ability to identify microorganisms and quantitate their contribution to biofilms in clinical infections. A more robust model of biofilm infection along with more accurate diagnosis is rapidly translating into improved clinical outcomes.

Chronic wounds and the medical biofilm paradigm.

There is a growing recognition that biofilms are the principal cause of wound chronicity. The development of treatments for wound biofilms raises the prospect that chronic wounds can be treated, potentially saving many patients' lives.

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

Specific anti cross-infection measures may help to prevent viral contamination of dental unit waterlines: a pilot study.

BACKGROUND: In recent years, several reports have suggested, but never definitely demonstrated that dental units (DU) could be potential sources of viral cross-infections sustained by viral agents including HBV, HCV and HIV. This work aims at assessing the risk of HCV cross-infection by dental unit water lines (DUWLs). MATERIALS AND METHODS: Ten anti-HCV positive viremic patients were submitted to dental treatment on three different DU (one unit fully equipped to minimize viral contamination risk). A PCR method using primers for UTR and E2 regions was used to evaluate HCV RNA presence in DUWLs sprays. A modified RNA extraction protocol was developed to eliminate the risk of low sensibility due to the presence of inhibitors in saliva. Sequences obtained from E2 PCR products amplified from blood and oral fluids were analyzed and compared. RESULTS: Fluids collected from three different DU before treatment were always negative for the presence of HCV RNA; after treatment viral contamination was detected in six out of ten cases in conventional DU, in three out of ten cases on the reduced-retraction DU while was never detected in sprays taken from fully equipped DU. Comparison of E2 region sequences obtained from blood and DUWLs sprays showed identity in each patient. CONCLUSION: Here we demonstrate that fixed DUWLs and handpieces can be contaminated by viral agents and become a vehicle of cross-infection and that a specific online active decontamination system developed for both handpieces and fixed waterlines can eliminate this risk.

Bacterial communications in implant infections: a target for an intelligence war.

The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called ""quorum-sensing"". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.

Phenotypic and functional characterization of Bacillus anthracis biofilms.

Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.

Using an efficient biofilm detaching agent: an essential step for the improvement of endoscope reprocessing protocols.

Biofilms develop inside endoscope channels even when valid endoscope reprocessing protocols are applied. The use of an efficient biocide is not sufficient if the channels are not cleaned thoroughly prior to disinfection. This study compared new anti-biofilm combinations of detachment promoting agents with a cleaning product in current use. Tests were performed using Teflon tubing and a contamination device that reproduces conditions that are prevalent during endoscopy. Products were subjected to static+brushing or dynamic treatments, and their ability to remove a preformed biofilm was assessed. The residual biofilm after treatment was assessed and compared with untreated controls. The percentage of surface covered by biofilm was measured after staining with crystal violet. Culturable bacteria levels were determined by plating the bacteria scraped from the tubing surface and counting the colony-forming units (CFU). Further tests were performed on actual endoscopes that had been contaminated artificially. Biofilm removal was confirmed by scanning electron microscopy. This study showed that the new anti-biofilm products prevented the build-up of biofilm and removed a mature biofilm (approximately 10(8)CFU/cm(2)), whereas protocols based on detergent-disinfectants containing quaternary ammonium compounds showed low efficacy as these protocols and products fixed the biofilm on the endoscope surfaces. The new procedure and agents represent a new approach to biofilm control that may improve the efficacy of endoscope reprocessing, and reduce the risk of transmitting infections.

Biofilm in implant infections: its production and regulation.

A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intraocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.A significant proportion of medical implants become the focus of a device-related infection, difficult to eradicate because bacteria that cause these infections live in well-developed biofilms. Biofilm is a microbial derived sessile community characterized by cells that are irreversibly attached to a substratum or interface to each other, embedded in a matrix of extracellular polymeric substances that they have produced. Bacterial adherence and biofilm production proceed in two steps: first, an attachment to a surface and, second, a cell-to-cell adhesion, with pluristratification of bacteria onto the artificial surface. The first step requires the mediation of bacterial surface proteins, the cardinal of which is similar to S. aureus autolysin and is denominated AtlE. In staphylococci the matrix of extracellular polymeric substances of biofilm is a polymer of beta-1,6-linked N-acetylglucosamine (PIA), whose synthesis is mediated by the ica operon. Biofilm formation is partially controlled by quorum sensing, an interbacterial communication mechanism dependent on population density. The principal implants that can be compromised by biofilm associated infections are: central venous catheters, heart valves, ventricular assist devices, coronary stents, neurosurgical ventricular shunts, implantable neurological stimulators, arthro-prostheses, fracture-fixation devices, inflatable penile implants, breast implants, cochlear implants, intra-ocular lenses, dental implants. Biofilms play an important role in the spread of antibiotic resistance. Within the high dense bacterial population, efficient horizontal transfer of resistance and virulence genes takes place. In the future, treatments that inhibit the transcription of biofilm controlling genes might be a successful strategy in inhibiting these infections.

Ultrasonically controlled release of ciprofloxacin from self-assembled coatings on poly(2-hydroxyethyl methacrylate) hydrogels for Pseudomonas aeruginosa biofilm prevention.

Indwelling prostheses and subcutaneous delivery devices are now routinely and indispensably employed in medical practice. However, these same devices often provide a highly suitable surface for bacterial adhesion and colonization, resulting in the formation of complex, differentiated, and structured communities known as biofilms. The University of Washington Engineered Biomaterials group has developed a novel drug delivery polymer matrix consisting of a poly(2-hydroxyethyl methacrylate) hydrogel coated with ordered methylene chains that form an ultrasound-responsive coating. This system was able to retain the drug ciprofloxacin inside the polymer in the absence of ultrasound but showed significant drug release when low-intensity ultrasound was applied. To assess the potential of this controlled drug delivery system for the targeting of infectious biofilms, we monitored the accumulation of Pseudomonas aeruginosa biofilms grown on hydrogels with and without ciprofloxacin and with and without exposure to ultrasound (a 43-kHz ultrasonic bath for 20 min daily) in an in vitro flow cell study. Biofilm accumulation from confocal images was quantified and statistically compared by using COMSTAT biofilm analysis software. Biofilm accumulation on ciprofloxacin-loaded hydrogels with ultrasound-induced drug delivery was significantly reduced compared to the accumulation of biofilms grown in control experiments. The results of these studies may ultimately facilitate the future development of medical devices sensitive to external ultrasonic impulses and capable of treating or preventing biofilm growth via "on-demand" drug release.

Can laboratory reference strains mirror "real-world" pathogenesis?

The extraordinary plasticity of bacterial genomes raises concerns about the adequacy of laboratory-adapted reference strains for the study of "real-world" pathogenesis. Some laboratory strains have been sub-cultured for decades since their first isolation and might have lost important pathophysiological characteristics. Evidence is presented that bacteria rapidly adapt to in vitro conditions. Genomic differences between laboratory reference strains and corresponding low-passage clinical isolates are reviewed. It appears that no bacterial strain can truly represent its species. For DNA microarray and proteomic studies, this limitation might be overcome by the summation of individual genomes to produce a species-specific virtual supragenome.

Survival strategies of infectious biofilms.

Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addition, biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. We review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiology of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell density, two key characteristics of biofilm physiology, also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.

Biofilm removal from silicone tubing: an assessment of the efficacy of dialysis machine decontamination procedures using an in vitro model.

The aim of this study was to assess the efficacy of 21 decontamination procedures, for the removal of a multispecies biofilm. Experiments were performed on five-day-old biofilms grown inside silicone tubing, using a reactor system that mimics a dialysis machine. The treatments were tested on 5 cm tubing samples. Effects of treatment were measured using direct microscopy following staining. Bacterial viability and endotoxin removal were determined using conventional microbiological methods following biofilm detachment by scraping. The 21 procedures were classified into four groups based on the amount of biofilm removed. The most effective treatment was an acid pre-treatment, followed by use of a concentrated bleach solution. Acid pre-treatment removes calcium and magnesium carbonate crystals that are always found in dialysis biofilms. Treatments performed at high temperature did not increase the efficacy of biofilm removal. Most treatments caused at least a 10(5)-fold reduction in bacterial viability with a few resulting in complete kill. Autoclaved and bleach-treated samples gave the best results for viability reduction, with both treatments providing an equally effective and complete kill. In addition, autoclaving led to a significant decrease in endotoxin level (removal of 99.99%).

Human leukocytes adhere to, penetrate, and respond to Staphylococcus aureus biofilms.

Staphylococcus aureus is a common pathogen responsible for nosocomial and community infections. It readily colonizes indwelling catheters, forming microbiotic communities termed biofilms. S. aureus bacteria in biofilms are protected from killing by antibiotics and the body's immune system. For years, one mechanism behind biofilm resistance to attack from the immune system's sentinel leukocytes has been conceptualized as a deficiency in the ability of the leukocytes to penetrate the biofilm. We demonstrate here that under conditions mimicking physiological shear, leukocytes attach, penetrate, and produce cytokines in response to maturing and fully matured S. aureus biofilm.

Influence of hydrodynamics and cell signaling on the structure and behavior of Pseudomonas aeruginosa biofilms.

Biofilms were grown from wild-type (WT) Pseudomonas aeruginosa PAO1 and the cell signaling lasI mutant PAO1-JP1 under laminar and turbulent flows to investigate the relative contributions of hydrodynamics and cell signaling for biofilm formation. Various biofilm morphological parameters were quantified using Image Structure Analyzer software. Multivariate analysis demonstrated that both cell signaling and hydrodynamics significantly (P < 0.000) influenced biofilm structure. In turbulent flow, both biofilms formed streamlined patches, which in some cases developed ripple-like wave structures which flowed downstream along the surface of the flow cell. In laminar flow, both biofilms formed monolayers interspersed with small circular microcolonies. Ripple-like structures also formed in four out of six WT biofilms, although their velocity was approximately 10 times less than that of those that formed in the turbulent flow cells. The movement of biofilm cell clusters over solid surfaces may have important clinical implications for the dissemination of biofilm subject to fluid shear, such as that found in catheters. The ability of the cell signaling mutant to form biofilms in high shear flow demonstrates that signaling mechanisms are not required for the formation of strongly adhered biofilms. Similarity between biofilm morphologies in WT and mutant biofilms suggests that the dilution of signal molecules by mass transfer effects in faster flowing systems mollifies the dramatic influence of signal molecules on biofilm structure reported in previous studies.

Immunology of Staphylococcal biofilm infections in the eye: new tools to study biofilm endophthalmitis.

Endophthalmitis is an important disease of the eye that is most frequently caused by postoperative and post-traumatic introduction of bacteria into the posterior segment of the eye. In the case of severe infections, visual acuity is greatly damaged or completely lost. Much work has focused on the ability of planktonic bacteria to cause infection and ocular damage while little work has focused on chronic infections in endophthalmitis mediated by the formation of bacterial biofilms on the surface of the lens. This review focuses on the interaction of Staphylococcus aureus and Staphylococcus epidermidis lens-associated biofilms in endophthalmitis. Additionally, this review highlights some relevant biofilm-immune system interactions and outlines a new in vivo mouse model to explore biofilm-related infections in endophthalmitis.

Biofilms as complex differentiated communities.

Prokaryotic biofilms that predominate in a diverse range of ecosystems are often composed of highly structured multispecies communities. Within these communities metabolic activities are integrated, and developmental sequences, not unlike those of multicellular organisms, can be detected. These structural adaptations and interrelationships are made possible by the expression of sets of genes that result in phenotypes that differ profoundly from those of planktonically grown cells of the same species. Molecular and microscopic evidence suggest the existence of a succession of de facto biofilm phenotypes. We submit that complex cell-cell interactions within prokaryotic communities are an ancient characteristic, the development of which was facilitated by the localization of cells at surfaces. In addition to spatial localization, surfaces may have provided the protective niche in which attached cells could create a localized homeostatic environment. In a holistic sense both biofilm and planktonic phenotypes may be viewed as integrated components of prokaryote life.

Antimicrobial activity of a novel catheter lock solution.

Intravascular catheter-associated bloodstream infections significantly increase rates of morbidity and hospital costs. Microbial colonization and development of biofilms, which are known to be recalcitrant to antibiotic therapy, often lead to the loss of otherwise patent vascular access systems. We evaluated a new taurolidine- and citrate-based catheter lock solution (Neutrolin; Biolink Corporation, Norwell, Mass.) for its activity against planktonic microbes, antimicrobial activity in a catheter model, and biofilm eradication activity. In studies of planktonic microbes, after 24 h of contact, 675 mg of taurolidine-citrate solution per liter caused > 99% reductions in the initial counts of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Entercoccus faecalis. A solution of 13,500 mg/liter was cidal for Candida albicans. Ports and attached catheters inoculated with 50 to 600 CFU of these bloodstream isolates per ml were locked with heparin or the taurolidine-citrate solution. After 72 h, there was no growth in the taurolidine-citrate-treated devices but the heparin-treated devices exhibited growth in the range of 6 x 10(2) to 5 x 10(6) CFU/ml. Biofilms were developed on silicone disks in modified Robbins devices with broth containing 6% serum (initial counts, 10(6) to 10(8) CFU/cm(2)). The axenic biofilms were treated for 24 h with taurolidine-citrate or heparin. Taurolidine-citrate exposure resulted in a median reduction of 4.8 logs, whereas heparin treatment resulted in a median reduction of 1.7 logs (P < 0.01). No significant differences in the effects of the two treatments against P. aeruginosa and C. albicans were observed. These findings suggest that taurolidine-citrate is a promising combination agent for the prevention and treatment of intravascular catheter-related infections.

Growth and detachment of cell clusters from mature mixed-species biofilms.

Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 microm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.