A novel protein-repellent dental composite containing 2-methacryloyloxyethyl phosphorylcholine

Secondary caries due to biofilm acids is a primary cause of dental composite restoration failure.To date,there have been no reports of dental composites that can repel protein adsorption and inhibit bacteria attachment.The objectives of this study were to develop a protein-repellent dental composite by incorporating 2-methacryloyloxyethyl phosphorylcholine(MPC) and to investigate for the first time the effects of MPC mass fraction on protein adsorption,bacteria attachment,biofilm growth,and mechanical properties.Composites were synthesized with 0(control),0.75%,1.5%,2.25%,3%,4.5%and 6%of MPC by mass.A commercial composite was also tested as a control.Mechanical properties were measured in three-point flexure.Protein adsorption onto the composite was determined by the microbicinchoninic acid method.A human saliva microcosm biofilm model was used.Early attachment at 4 h,biofilm at 2 days,live/dead staining and colony-forming units(CFUs) of biofilms grown on the composites were investigated.Composites with MPC of up to 3%had mechanical properties similar to those without MPC and those of the commercial control,whereas 4.5%and 6%MPC decreased the mechanical properties(P<0.05).Increasing MPC from 0 to 3%reduced the protein adsorption on composites(P<0.05).The composite with 3%MPC had protein adsorption that was 1/12 that of the control(P<0.05).Oral bacteria early attachment and biofilm growth were also greatly reduced on the composite with 3%MPC,compared to the control(P<0.05).In conclusion,incorporation of MPC into composites at 3%greatly reduced protein adsorption,bacteria attachment and biofilm CFUs,without compromising mechanical properties.Protein-repellent composites could help to repel bacteria attachment and plaque build-up to reduce secondary caries.The protein-repellent method might be applicable to other dental materials.     

In vitro analysis of riboflavin-modified,experimental, two-step etch-and-rinse dentin adhesive:Fourier transform infrared spectroscopy and micro-Raman studies

To modify two-step experimental etch-and-rinse dentin adhesive with different concentrations of riboflavin and to study its effect on the bond strength,degree of conversion,along with resin infiltration within the demineralized dentin substrate,an experimental adhesive-system was modified with different concentrations of riboflavin(mlm,0,1%,3%,5%and 10%).Dentin surfaces were etched with 37%phosphoric acid,bonded with respective adhesives,restored with restorative composite-resin,and sectioned into resin-dentin slabs and beams to be stored for 24 h or 9 months in artificial saliva.Micro-tensile bond testing was performed with scanning electron microscopy to analyse the failure of debonded beams.The degree of conversion was evaluated with Fourier transform infrared spectroscopy(FTIR) at different time points along with micro-Raman spectroscopy analysis.Data was analyzed with one-way and two-way analysis of variance followed by Tukey’s for pair-wise comparison.Modification with 1%and 3%riboflavin increased the micro-tensile bond strength compared to the control at 24 h and 9-month storage with no significant differences in degree of conversion(P<0.05).The most predominant failure mode was the mixed fracture among all specimens except 10%riboflavin-modified adhesive specimens where cohesive failure was predominant.Raman analysis revealed that 1%and 3%riboflavin adhesives specimens showed relatively higher resin infiltration.The incorporation of riboflavin in the experimental two-step etch-and-rinse adhesive at 3%(mlm) improved the immediate bond strengths and bond durability after 9-month storage in artificial saliva without adversely affecting the degree of conversion of the adhesive monomers and resin infiltration.     

Effects of quaternary ammonium chain length on the antibacterial and remineralizing effects of a calcium phosphate nanocomposite

Composites containing nanoparticles of amorphous calcium phosphate (NACP) remineralize tooth lesions and inhibit caries. A recent study synthesized quaternary ammonium methacrylates (QAMs) with chain lengths (CLs) of 3–18 and determined their effects on a bonding agent. This study aimed to incorporate these QAMs into NACP nanocomposites for the first time to simultaneously endow the material with antibacterial and remineralizing capabilities and to investigate the effects of the CL on the mechanical and biofilm properties. Five QAMs were synthesized: DMAPM (CL3), DMAHM (CL6), DMADDM (CL12), DMAHDM (CL16), and DMAODM (CL18). Each QAM was incorporated into a composite containing 20% NACP and 50% glass fillers. A dental plaque microcosm biofilm model was used to evaluate the antibacterial activity. The flexural strength and elastic modulus of nanocomposites with QAMs matched those of a commercial control composite (n 5 6; P . 0.1). Increasing the CL from 3 to 16 greatly enhanced the antibacterial activity of the NACP nanocomposite (P , 0.05); further increasing the CL to 18 decreased the antibacterial potency. The NACP nanocomposite with a CL of 16 exhibited biofilm metabolic activity and acid production that were 10-fold lesser than those of the control composite. The NACP nanocomposite with a CL of 16 produced 2-log decreases in the colony-forming units (CFU) of total microorganisms, total streptococci, and mutans streptococci. In conclusion, QAMs with CLs of 3–18 were synthesized and incorporated into an NACP nanocomposite for the first time to simultaneously endow the material with antibacterial and remineralization capabilities. Increasing the CL reduced the metabolic activity and acid production of biofilms and caused a 2-log decrease in CFU without compromising the mechanical properties. Nanocomposites exhibiting strong anti-biofilm activity, remineralization effects, and mechanical properties are promising materials for tooth restorations that inhibit caries.     

Antimicrobial activity of alexidine alone and associated with N-acetylcysteine against Enterococcus faecalis biofilm

The purpose of this study was to assess the efficacy of alexidine（ALX）,alone and combined with N-acetylcysteine（NAC）,in eradicating two Enterococcus faecalis strain biofilms.The biofilms of E.faecalis ATCC 29212 and the clinical isolate E.faecalis D1 were grown in the MBEC-high-throughput device for 24 h and were exposed to five twofold dilutions of ALX（2%–0.007 8%）alone and combined with100 mg？mL21NAC,for 1 and 5 min.Eradication was defined as 100%kill of biofilm bacteria.The Student’s t-test was used to compare the efficacy of the associations of the two irrigants.After 1-min contact time,ALX eradicated the biofilms at all concentrations except for 0.007 8%and 0.015 6%–0.007 8%with E.faecalis ATCC 29212 and E.faecalis D1,respectively.Similar results for eradication and concentration were obtained when it was combined with 100 mg？mL21NAC.After 5 min of contact time,ALX alone and combined with NAC eradicated all enterococci biofilms.ALX showed antimicrobial properties against the two E.faecalis strain biofilms tested at very low concentrations,and its combined use with NAC was not seen to enhance its activity.     

Gentian violet and ferric ammonium citrate disrupt Pseudomonas aeruginosa biofilms

Objective/Hypothesis: Bacterial biofilms are resistant to antibiotics and may contribute to persistent infections including chronic otitis media and cholesteatoma. Discovery of substances to disrupt biofilms is necessary to treat these chronic infections. Gentian violet (GV) and ferric ammonium citrate (FAC) were tested against Pseudomonas aeruginosa biofilms to determine if either substance can reduce biofilm volume. Study Design: The biofilm volume and planktonic growth of PAO1 and otopathogenic P. aeruginosa (OPPA8) isolated from an infected cholesteatoma was measured in the presence of GV or FAC. Methods: OPPA8 and PAO1 expressing a green fluorescent protein plasmid (pMRP9‐1) was inoculated into a glass flow chamber. Biofilms were grown under low flow conditions for 48 hours and subsequently exposed to either GV or FAC for an additional 24 hours. Biofilm formation was visualized by confocal laser microscopy and biofilm volume was assayed by measuring fluorescence. Planktonic cultures were grown under standard conditions with GV or FAC. Statistical analysis was performed by Student t test and one‐way ANOVA. Results: GV reduced PAO1 and OPPA8 biofilm volume (P < .01). GV delayed the onset and rate of logarithmic growth in both strains. FAC reduced OPPA8 biofilm volume (P < .01), but did not effect of PAO1 biofilms. FAC had no effect on planktonic growth. Conclusions: The efficacy of GV in disrupting biofilms in vitro suggests that it may disrupt biofilms in vivo. The effect of FAC on Pseudomonas aeruginosa biofilms is strain dependent. Strain differences in response to increasing iron concentration and biofilm morphology stress the importance of studying clinically isolated strains in testing antibiofilm agents.     

Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection

Aim Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. Mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. Mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. Mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent 5. Mutans colonization. Methodology Controlled amounts of S. Mutans were mixed with S. Mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. Mutans were tested for their ability to reduce S. Mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their abilityto resist subsequent exogenous S. Mutans colonization. Results S. Mutans colonization was considerably reduced (9 ±0.4 fold reduction, P=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. Mutans-specific STAMPs yielded S. Mutans-deficient biofilms with significant protection against further S. Mutans colonization (5 minutes treatment: 38 ± 13 fold reduction P=0.01; 16 hours treatment: 96 ±28 fold reduction P=0.07).Conclusion S. Mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or "normal" biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. Mutans colonization in the oral cavity and caries progression.     

Characterization of mucosal biofilms on human adenoid tissues

OBJECTIVES: To demonstrate the presence of mucosal biofilm in adenoid tissue using double staining for visualization of both the bacterial matrix and the bacterial cells. To identify bacterial species present on the surface of the studied adenoids. STUDY DESIGN: Prospective study. METHODS: A total of 39 specimens of adenoidectomy were removed from children with chronic and/or recurrent otitis media. The specimens were prepared for light microscopy using Gram staining, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Double staining was performed with CLSM to visualize both the bacteria and the glycocalyx matrix. Nine adenoids on which bacterial biofilms were visualized with CLSM were used for identification of bacterial species by 16S-DNA polymerase chain reaction (PCR) amplification and homology analysis. RESULTS: Of the 39 adenoids investigated, 22 (54%) showed evidence of mucosal biofilms. Gram staining, SEM and CLSM showed the presence of bacterial cells, organized in bacterial microcolonies. CLSM with double staining demonstrated mucosal biofilms by showing the presence of both bacteria and the glycocalyx. The use of 16S-DNA polymerase chain reaction (PCR) amplification and subsequent sequence analyses identified the presence of Corynebacterium argentoratense, Streptococcus salivarius, Micrococcus luteus, and Staphylococcus aureus. CONCLUSIONS: This study demonstrates that adenoid tissue in children with chronic or/and recurrent otitis media contains mucosal biofilms in 54% of the cases. The existence of living bacteria has been demonstrated. Further studies are required to describe the panel of bacteria that can be harbored in the biofilms present in adenoids and the mechanisms involved in the physiopathology of otitis prone children.     

Primer containing dimethylaminododecyl methacrylate kills bacteria impregnated in human dentin blocks

Antibacterial dimethylaminododecyl methacrylate (DMADDM) was recently synthesized. The objectives of this study were to:(1) investigate antibacterial activity of DMADDM-containing primer on Streptococcus mutans impregnated into dentin blocks for the first time, and (2) compare the antibacterial efficacy of DMADDM with a previous quaternary ammonium dimethacrylate (QADM). Scotchbond Multi-Purpose (SBMP) bonding agent was used. DMADDM and QADM were mixed into SBMP primer. Six primers were tested:SBMP control primer P, P+2.5%DMADDM, P+5%DMADDM, P+7.5%DMADDM, P+10%DMADDM, and P+10%QADM. S. mutans were impregnated into human dentin blocks, and each primer was applied to dentin to test its ability to kill bacteria in dentinal tubules. Bacteria in dentin were collected via a sonication method, and the colony-forming units (CFU) and inhibition zones were measured. The bacterial inhibition zone of P+10%DMADDM was 10 times that of control primer (Po0.05). CFU in dentin with P+10%DMADDM was reduced by three orders of magnitude, compared with control. DMADDM had a much stronger antibacterial effect than QADM, and antibacterial efficacy increased with increasing DMADDM concentration. Dentin shear bond strengths were similar among all groups (P40.1). In conclusion, antibacterial DMADDM-containing primer was validated to kill bacteria inside dentin blocks, possessing a much stronger antibacterial potency than the previous QADM. DMADDM-containing bonding agent was effective in eradicating bacteria in dentin, and its efficacy was directly proportional to DMADDM mass fraction. Therefore, DMADDM may be promising for use in bonding agents as well as in other restorative and preventive materials to inhibit bacteria.     

Evidence for microbial biofilms in cholesteatomas

BACKGROUND: Sessile bacteria within biofilms are highly resistant to eradication by antimicrobial agents. Previously, we have shown that the most common organisms cultured from experimentally induced cholesteatomas are biofilm formers. Additionally, the keratin "matrix" of a cholesteatoma is an ideal environment for the support of biofilm formation. OBJECTIVE: To determine if microbial biofilms occur within the keratin matrix of infected cholesteatomas. DESIGN: We evaluated the histomorphologic characteristics of 24 human and 22 experimental cholesteatomas for evidence of biofilm formation using light and transmission electron microscopy. SUBJECTS: Human tissues were collected during surgical eradication of existing cholesteatomas. Twenty-two gerbil cholesteatomas were either spontaneously occurring or induced by external auditory canal ligation and harvested several months later. RESULTS: Gram-positive and gram-negative bacteria were seen within acellular deposits among the keratin accumulations in 21 of 22 gerbil and 16 of 24 human cholesteatomas. Regions of accumulated bacteria possessed the ultrastructural appearance of typical amorphous polysaccharide biofilm matrix. CONCLUSIONS: There is strong anatomic evidence for the presence of bacterial biofilms in experimental and human cholesteatomas. The existence of bacterial biofilms within cholesteatomas may explain the clinical characteristics of infected cholesteatomas, that is, persistence and recurrence of infection, with surgical eradication being the only effective treatment.     

Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate（BP）-related osteonecrosis of the jaw（ONJ） or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts（n530）; patients with periodontal disease without a history of BP therapy（Control, n510）, patients with periodontal disease having history of BP therapy but without ONJ（BP, n55） and patients with BRONJ（BRONJ, n515）. Denaturing gradient gel electrophoresis of polymerase chain reaction（PCR）-amplified 16 S r RNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ（71.6%）, BP（70.3%） and Control（59.1%）. Significant differences（P,0.05） in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay（ELISA）results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix–loop–helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.     

Bacterial biofilms in surgical specimens of patients with chronic rhinosinusitis

OBJECTIVES: Biofilms are bacterial pathogens that organize in several chronic and recalcitrant infectious processes. We hypothesize that biofilms play a role in chronic rhinosinusitis (CRS). Our goal is to demonstrate biofilms in mucosal specimens of patients undergoing surgery for CRS. STUDY DESIGN: A prospective study of the presence of biofilms in patients undergoing endoscopic sinus surgery for CRS compared with control patients without CRS. METHODS: There were a total of 30 subjects and 4 controls enrolled. The samples of 24 subjects and 4 controls were cultured and then prepared using standard methods for scanning electron microscopy (SEM). The remaining six subjects' samples were treated using advanced cryofixation methods as preparation to preserve structure for SEM and transmission electron microscopy (TEM). RESULTS: Using strict SEM morphologic criteria, 24 (80%) of the 30 patients were found to have micrographic evidence of biofilms. All controls had healthy appearing cilia and goblet cells without biofilms. The six cryofixation samples showed biofilm structures on SEM micrographs that were correlated with bacterial structures seen at the mucosal surface on the corresponding TEM cross sections. Bacterial cultures were positive on all patients. CONCLUSIONS: Biofilms were demonstrated to be present in patients undergoing surgery for CRS; none of the patients without CRS had any evidence of biofilms. Although SEM is capable of demonstrating the biofilms' three-dimensional structure, glycocalyx, and water channels, it cannot clearly demonstrate the presence of bacteria within the biofilm. We were able to demonstrate evidence of bacteria in the biofilms on the subjects tested using TEM.     

The clinical impact of bacterial biofilms

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.     

One-year water-ageing of calcium phosphate composite containing nano-silver and quaternary ammonium to inhibit biofilms

Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were (1) to develop a composite containing quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to conduct the first investigation of the mechanical properties, biofilm response and acid production vs water-ageing time from 1 day to 12 months. A 4 × 5 design was utilized, with four composites (NACP-QADM composite, NACP-NAg composite, NACP-QADM-NAg composite, and a commercial control composite), and five water-ageing time periods (1 day, and 3, 6, 9, and 12 months). After each water-ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM-NAg had a flexural strength and elastic modulus matching those of the commercial control (P40.1). Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone (Po0.05). The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time (P40.1). In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load-bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.     

Bacterial biofilms on the sinus mucosa of human subjects with chronic rhinosinusitis

INTRODUCTION: Chronic rhinosinusitis (CRS) is a common disease poorly controlled by antibiotics. Postulated etiologies of CRS include allergy, fungi, functional factors, and biofilm. OBJECTIVES: We presented a preliminary study demonstrating bacterial biofilms' presence on the sinus mucosa of patients with CRS using fluorescent in situ hybridization (FISH). The advantage of FISH in biofilm identification is that it is the only method that identifies the specific bacteria creating the biofilm matrix. We now present the results of a larger series of patients. METHODS: Patients with CRS scheduled for sinus surgery were enrolled in the study. Biopsies of the sinus mucosa and cultures were taken at the time of surgery. Control samples were taken from patients undergoing septoplasty. Specimens underwent FISH testing for Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenza, and Pseudomonas aeruginosa. RESULTS: Bacterial biofilms were present on 14 of 18 specimens. The predominant species were H. influenzae, S. pneumoniae, and S. aureus. P. aeruginosa biofilm was not identified on any specimens. The intraoperative cultures of the planktonic bacteria present in the sinuses did not correlate with the biofilms identified. Two of the five control samples were positive for biofilm. CONCLUSION: The presence of biofilms on the mucosa of patients with CRS offers a possible cause of antimicrobial therapy failure and could change the approach to treatment. However, the presence of biofilms on healthy control samples implies that biofilms may simply be colonizers. The precise role that biofilms play in CRS still remains to be determined. Further studies with larger sample sizes are needed.     

Multispecies communities: interspecies interactions influence growth on saliva as sole nutritional source

Human oral bacteria live in multispecies communities in the biofilm called dental plaque.This review focuses on the interactions of seven species and the ability of each species individually and together with other species to grow on saliva as the sole source of nutrient.Community formation in biofilms in flow cells is monitored using species-specific fluorophore-conjugated immunoglobulin G,and images are captured by confocal microscopy.Early colonizing veillonellae emerge from this review of interspecies interactions in saliva as a critical genus that guides the development of multispecies communities.Highly selective interspecies recognition is evident as initial colonizers pair with early and middle colonizers to form multispecies communities that grow on saliva.     

Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media

BACKGROUND: An increased awareness of bacterial biofilms and their formation has led to a better understanding of bacterial infections that occur in the middle ear. Perhaps the best studied pathogen for its propensity toward biofilm formation is Pseudomonas aeruginosa, also the primary pathogen in chronic suppurative otitis media (CSOM). OBJECTIVE: The aim of this study was to determine whether P. aeruginosa forms a biofilm in the middle ear in the setting of CSOM in a nonhuman primate model. METHODS: Cynomolgus monkeys underwent perforation of the tympanic membrane and inoculation of the middle ear with a known biofilm-forming strain of P. aeruginosa. The contralateral ear was used as an internal control and was neither perforated nor infected. At the end of the study period, both ears were irrigated to remove planktonic bacteria, and the middle ear mucosa was removed and examined ultrastructurally using scanning electron microscopy (SEM) for determination of the presence or absence of biofilm formation. MAIN OUTCOME MEASURE: The identification of middle ear biofilm containing rod-shaped bacteria. RESULTS: SEM revealed that P. aeruginosa formed bacterial biofilm in vivo on the middle ear mucosal surface, seen only in the infected ear. Interestingly, biofilm formation caused by cocci was also seen in both the experimental as well as the control ear. CONCLUSION: P. aeruginosa forms biofilms in the middle ear in CSOM in primates. To our knowledge, this is the first report of disease-associated bacterial biofilm in a nonhuman primate model of CSOM. Such a model lays a foundation for much needed study into the role of biofilms in the pathophysiology of CSOM. Should CSOM be caused by biofilms, which is uncertain at this time, development of novel strategies for treatment and prevention may be possible. The finding of both rods and cocci forming biofilms also warrants further investigation.     

Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries

Alkali production by oral bacteria is believed to have a major impact on oral microbial ecology and to be inibitory to the initiation and progression of dental caries.A substantial body of evidence is beginning to accumulate that indicates the modulation of the alkalinogenic potential of dental biofilms may be a promising strategy for caries control.This brief review highlights recent progress toward understanding molecular genetic and physiologic aspects of important alkali-generating pathways in oral bacteria,and the role of alkali production in the ecology of dental biofilms in health and disease.     

Direct evidence of bacterial biofilms in otitis media

OBJECTIVES/HYPOTHESIS: Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations. STUDY DESIGN: Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients. METHODS: Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in children's ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes. RESULTS: Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from children's ears for clinical reasons, in contrast with unused control tubes. CONCLUSIONS: Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in children's ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.     

Determination of the biofilm formation capacity of bacterial pathogens associated with otorhinolaryngologic diseases in the Malaysian population

This study aims to assess the association between microbial composition, biofilm formation and chronic otorhinolaryngologic disorders in Malaysia. A total of 45 patients with chronic rhinosinusitis, chronic tonsillitis and chronic suppurative otitis media and 15 asymptomatic control patients were studied. Swab samples were obtained from these subjects. Samples were studied by conventional microbiological culturing, PCR-based microbial detection and Confocal Laser Scanning Microscopy (CLSM). Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative staphylococci (CoNS) and other Streptococcus species were detected in subjects of both patient and control groups. Biofilm was observed in approximately half of the smear prepared from swab samples obtained from subjects of the patient group. Most of these were polymicrobial biofilms. S. aureus biofilm was most prevalent among nasal samples while H. influenzae biofilm was more common among ear and throat samples. Results from this study supported the hypothesis that chronic otorhinolaryngologic diseases may be biofilm related. Due to the presence of unculturable bacteria in biofilms present in specimens from ear, nose and throat, the use of molecular methods in combination with conventional microbiological culturing has demonstrated an improvement in the detection of bacteria from such specimens in this study.