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.

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.

Bacterial biofilms: a common cause of persistent infections.

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. Formation of these sessile communities and their inherent resistance to antimicrobial agents are at the root of many persistent and chronic bacterial infections. Studies of biofilms have revealed differentiated, structured groups of cells with community properties. Recent advances in our understanding of the genetic and molecular basis of bacterial community behavior point to therapeutic targets that may provide a means for the control of biofilm infections.

The involvement of cell-to-cell signals in the development of a bacterial biofilm.

Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically and physiologically differentiated from free-living bacteria. A cell-to-cell signal is involved in the development of Pseudomonas aeruginosa biofilms. A specific signaling mutant, a lasI mutant, forms flat, undifferentiated biofilms that unlike wild-type biofilms are sensitive to the biocide sodium dodecyl sulfate. Mutant biofilms appeared normal when grown in the presence of a synthetic signal molecule. The involvement of an intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in cystic fibrosis, and in other environments where P. aeruginosa biofilms are a persistent problem.

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.

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.

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.

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.

Cystic fibrosis pathogenesis and the role of biofilms in persistent infection.

Recent work has presented definitive evidence that the Pseudomonas cells that infect the lung in cystic fibrosis grow in the biofilm phenotype. These unequivocal data establish this chronic infection of compromised hosts as the archetypal biofilm infection, which is both refractory to antibiotic therapy and barely affected by host defenses.

A scanning electron microscopic study of urine droppers and urine collecting systems.

In this study we have shown extensive microbial colonization of the surface of urine droppers and urine collecting systems from bacteriuric patients. In all instances there was an extensive background matrix and fibrous strands interconnected the bacteria. A "slime" layer completely encased bacteria on occasion. These observations suggest that incorporation of blockers of bacterial adhesion into the surfaces of these devices should be explored as one method of delaying, or in some cases preventing, catheter-acquired bacteriuria.

Adherent microorganisms on lumenal surfaces of long-term intravenous catheters. Importance of Staphylococcus epidermidis in patients with cancer.

Using electron microscopy, we prospectively evaluated how frequently adherent microorganisms colonized silicone rubber intravenous (Hickman) catheters removed from patients with cancer. Thirteen (87%) of 15 catheters had gram-positive cocci in glycocalyx adherent to the surface of the catheter lumen. Fungal elements or gram-negative bacilli were mixed with the gram-positive cocci in the glycocalyx on the lumens of three catheters. A consistent morphologic form was adherent to, and the same species was recovered from, the corresponding catheter for six of 27 organisms causing septicemia during catheterization: four of five Staphylococcus epidermidis bacteremias and the only Staphylococcus aureus bacteremia, and one of five candidemias. Three of these six septicemias were successfully treated without removal of the catheter. Although adherent organisms, particularly S epidermidis, were likely to be present on the surface of the lumen of long-term, indwelling, silicone intravenous catheters, septicemias potentially related to these organisms occurred infrequently (fewer than two per 1000 days of catheter use), and the suspect septicemias could sometimes be treated without removal of the catheter.

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.

Therapeutic efficacy of fleroxacin for eliminating catheter-associated urinary tract infection in a rabbit model.

The efficacy of fleroxacin as therapy for experimentally induced catheter-associated urinary tract infection (CAUTI) was examined. A rabbit model of CAUTI using a closed urinary catheter drainage system and the mutant strain of Escherichia coli (WE 6933) were used to examine three dosage regimens (30 mg/kg q8h i.v.; 20 mg/kg q8h i.v.; and 10 mg/kg q8h++i.v.) of fleroxacin administered intravenously for 4 days. Quantitative bacterial counts, urinary concentrations of fleroxacin and desmethylferoxacin, histopathologic changes, and electron microscopic evaluation of catheter-associated biofilm and mucosal biofilm were performed. The results indicated that the bacterial biofilm on the urinary catheter could be eliminated by fleroxacin at 30 mg/kg q8h i.v. and 20 mg/kg q8h i.v. Fleroxacin concentrations in urine exceeded the levels necessary to destroy E. coli. Viable bacteria were eliminated with the third regimen (10 mg/kg q8h i.v.), but electron microscopy demonstrated remnants of bacterial biofilm. Histopathologic changes were significantly reduced in all fleroxacin-treated rabbits, and scanning electron microscopy showed deterioration of the bacterial biofilm on the surface of the Foley catheter in treated animals. These data suggest that fleroxacin may be useful for treating catheter-related infections because these therapeutic dosages limited ascending infections of the urethra and bladder, eliminated catheter-associated biofilms, and killed planktonic bacteria in urine.

Guidewire catheter change in central venous catheter biofilm formation in a burn population.

This study was designed to assess the risk of colonization and biofilm formation of central venous catheters left in situ for seven days vs those changed over a guidewire at three days and removed at seven days. Colonization was determined using scanning and transmission electron microscopy and compared to a special scraping/sonication culture method. Thirty-one catheters were examined, and no difference was found between catheters left in situ (9 of 16 colonized) and those changed over a guidewire (11 of 15 colonized). Colonization rates rose significantly from 4 of 15 catheters at the time of guidewire change to 11 of 15 at 7 days (p less than 0.001). Of the catheters defined as colonized by SEM, the special culture technique showed bacterial growth in only 35 percent, making a negative culture result of dubious value in ruling out catheter colonization. No beneficial effect of guidewire changes in reducing colonization could be demonstrated.

Biofilms on right heart flow-directed catheters.

This study was designed to detect biofilm and bacteria on right heart flow-directed catheters using scanning electron microscopy and culture following scraping and dispersion of biofilm by sonication. We examined 20 consecutive catheters removed from 18 critically ill patients, an average of 2.6 days after insertion. On scanning electron microscopy, all catheters were found to be covered by a biofilm, with bacteria visible on 50 percent of them. Cultures of specimens from 40 percent of the catheters grew skin organisms (Staphylococcus warneri, Diphtheroid), anaerobes (Propionibacterium), and other potential pathogens (Proteus vulgaris, Enterobacter cloacae). Combination of the two techniques produced a bacterial detection rate of 75 percent. This study demonstrates that the presence of biofilm with bacterial adherence is common on right heart flow-directed catheters. The phenomenon could play a significant role in endogenous infection in critically ill patients.

Some bacterial parameters influencing the neutrophil oxidative burst response to Pseudomonas aeruginosa biofilms.

Persistence of bacteria in spite of a normal host immune system and relevant antibiotic treatment is a key problem in many chronic infections, such as the bronchopulmonary P. aeruginosa infection in cystic fibrosis patients. The capability of bacteria to establish themselves in microcolonies or biofilms is an important protective mechanism of the microorganisms. We examined the human PMN oxidative burst response to P. aeruginosa in biofilm and in planktonic form. The PMN chemiluminescence response to P. aeruginosa in biofilms was reduced to 30.5-47.5% (p less than 0.04) and the superoxide response to 85.9% (p less than 0.02) of the response to equivalent numbers of planktonic bacteria. Mechanical disruption of the biofilms before the assays elicited a significantly increased response in the chemiluminescence experiments and to nonopsonized biofilms in the superoxide anion experiments. We conclude that biofilm bacteria, although able to stimulate the PMN, result in a reduced, suboptimal response leading to lack of efficient eradication of the bacteria in the chronic infection.

Legionella pneumophila grows adherent to surfaces in vitro and in situ.

Legionella pneumophila continues to play a role in both community- and nosocomially-acquired pneumonia. We investigated the ability of L pneumophila to adhere to various types of materials such as those found in the hospital air-cooling and portable water distribution systems. Through the use of a unique sampling apparatus, we were able to regularly acquire planktonic and sessile samples and determine the numbers of bacteria present in both populations, in vitro and in situ. Portions of these apparatuses could be aseptically removed for examination by scanning electron microscopy, or for the determination of the number of viable adherent L pneumophila. The number of bacteria present in each sample was determined by direct plate count, with presumptive L pneumophila colonies being positively identified by direct fluorescent antibody staining techniques. The results demonstrated that not only are legionellae capable of colonizing various metallic and nonmetallic surfaces but that they are preferentially found on surfaces. Surface-adherent bacteria may play a profound role as a reservoir of these potential pathogens in aquatic environments. Furthermore, these results suggest that any comprehensive legionella monitoring program must include not only water samples but also an examination of the adherent populations.

Possible association of mucous blanket integrity with postirradiation colonization resistance.

Radiation-induced infections can be associated with changes in colonization potential of the intestine. Since the mucous blanket, which overlays the epithelium, is a major mucosal structure and is heavily colonized by microorganisms, we examined the status of the mucus after radiation and evaluated susceptibility to intestinal challenge with bacteria. A downward shift (2.5 X 10(8) cells/g to 5.3 X 10(5)) of total facultatively anaerobic bacteria of the ileum of C3HeB/FeJ mice was detected by 3 days post exposure to 10 Gy 60Co. Numbers of flora returned to normal by 11 days after radiation. Scanning electron microscopy was used to show that the loss of bacteria could be associated with major disruptions of the continuity of the mucous blanket. The pathogen Pseudomonas aeruginosa adhered to mouse mucous films used in in vitro assays. When irradiated mice were challenged orally with 1 X 10(5) P. aeruginosa on days 1, 2, or 3 after irradiation, a progressive increase in susceptibility was seen, but no animals died before Day 4 postirradiation. Sensitivity to subcutaneous (sc) challenge with Pseudomonas also increased by Day 3 and was probably due largely to the profound neutropenia observed. Immunoglobulin G (Gamimmune), which protected burned mice infected with Pseudomonas, was ineffectual in treatment of 7 or 10 Gy irradiated mice challenged either orally or sc with the organism. The ileal mucosal barrier was compromised after radiation in ways which could facilitate epithelial colonization, an event which combined with other immunological and physiological decrements in this model can compromise the effectiveness of therapeutic modalities.

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.