The influence of antimicrobial peptides and mucolytics on the integrity of biofilms consisting of bacteria and yeasts as affecting voice prosthetic air flow resistances

The integrity of biofilms on voice prostheses used to rehabilitate speech in laryngectomized patients causes unwanted increases in airflow resistance, impeding speech. Biofilm integrity is ensured by extracellular polymeric substances (EPS). This study aimed to determine whether synthetic salivary peptides or mucolytics, including N-acetylcysteine and ascorbic acid, influence the integrity of voice prosthetic biofilms. Biofilms were grown on voice prostheses in an artificial throat model and exposed to synthetic salivary peptides, mucolytics and two different antiseptics (chlorhexidine and Triclosan). Synthetic salivary peptides did not reduce the air flow resistance of voice prostheses afterm biofilm formation. Although both chlorhexidine and Triclosan reduced microbial numbers on the prostheses, only the Triclosan-containing positive control reduced the air flow resistance. Unlike ascorbic acid, the mucolytic N-acetylcysteine removed most EPS from the biofilms and induced a decrease in air flow resistance.     

Influence of different combinations of bacteria and yeasts in voice prosthesis biofilms on air flow resistance

Laryngectomized patients use silicone rubber voice prostheses to rehabilitate their voice. However, biofilm formation limits the lifetime of voice prostheses. The presence of particular combinations of bacterial and yeast strains in voice prosthesis biofilms has been suggested to be crucial for causing valve failure. In order to identify combinations of bacterial and yeast strains causative to failure of voice prostheses, the effects of various combinations of bacterial and yeast strains on air flow resistances of Groningen button voice prostheses were determined. Biofilms were grown on Groningen button voice prostheses by inoculating so-called artificial throats with various combinations of clinically relevant bacterial and yeast strains. After 3 days, all throats were perfused three times daily with 250 ml phosphate buffered saline and at the end of each day the artificial throats were filled with growth medium for half an hour. After 7 days, the air flow resistances of the prostheses were measured. These air flow resistances were expressed relative to the air flow resistances of the same prostheses prior to biofilm formation. This study shows that biofilms causing strong increases in air flow resistance (26 to 28 cm water.s/l) comprised combinations of microorganisms, involving Candida tropicalis, Staphylococcus aureus and Rothia dentocariosa. This revised version was published online in June 2006 with corrections to the Cover Date.     

Adhesive interactions between voice prosthetic yeast and bacteria on silicone rubber in the absence and presence of saliva

Biofilms on silicone rubber voice prostheses are the major cause for frequent failure and replacement of these devices. The presence of both bacterial strains and yeast has been suggested to be crucial for the development of voice prosthetic biofilms. Adhesive interactions between Candida albicans, Candida krusei, and Candida tropicalis with 14 bacterial strains, all isolated from explanted voice prostheses were investigated in a parallel plate flow chamber. Bacteria were first allowed to adhere to silicone rubber, after which the flow chamber was perfused with yeast, suspended either in saliva or buffer. Generally, when yeast were adhering from buffer and saliva, the presence of adhering bacteria suppressed adhesion of yeast. In saliva, Rothia dentocariosa and Staphylococcus aureus enhanced adhesion of yeast, especially of C. albicans. This study shows that bacterial adhesion mostly reduces subsequent adhesion of yeast, while only a few bacterial strains stimulate adhesion of yeast, provided salivary adhesion mediators are present. Interestingly, different clinical studies have identified R. dentocariosa and S. aureus in biofilms on explanted prostheses of patients needing most frequent replacement, while C. albicans is one of the yeast generally held responsible for silicone rubber deterioration.     

Caffeinated soft drinks reduce bacterial prevalence in voice prosthetic biofilms

Laryngectomized patients use indwelling silicone rubber voice prostheses, placed in a surgically created fistula in between the trachea and the esophagus, for voice and speech rehabilitation. At the esophageal side, these voice prostheses rapidly become colonized by a thick biofilm consisting of a variety of oral and skin bacteria and yeasts, and on average, after 3–4 months a prosthesis has to be replaced. In this study, the influence of caffeinated soft drinks on biofilm formation on silicone rubber voice prostheses has been investigated in a modified Robbins device. Robbins devices were first inoculated with the total cultivable microflora from an explanted voice prosthesis for 3 d, after which the devices were perfused three times daily over a 12 day period with 650 ml of either phosphate buffered saline or carbonated mineral water (controls), caffeinated soft drinks (two types), or a decaffeinated and a sugar‐free version of one of the caffeinated soft drinks. At the end of a day, during the experimental period, the devices were filled with growth medium for 30 min. Both caffeinated soft drinks reduced bacterial prevalence in the biofilms to 1–5% of the control, while yeasts thrived in voice prosthetic biofilms exposed to caffeinated soft drinks. Neither the controls, nor the decaffeinated soft drink, nor the sugar‐free version of this showed these effects on bacterial prevalence.     

Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.     

Voice Prosthetic Biofilm Formation and Candida Morphogenic Conversions in Absence and Presence of Different Bacterial Strains and Species on Silicone-Rubber

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.     

Mucolytics and antioxidant activity

We investigated effects of the mucolytics ambroxol and N-acetylcysteine on airways reactivity evoked by histamine in guinea pigs exposed to toluene vapors. We did not find significant changes in reactivity of tracheal smooth muscle from animals treated with mucolytics compared to the control group. However, the administration of ambroxol and N-acetylcysteine caused a significant decrease in lung tissue reactivity. The effect of ambroxol was more pronounced after intraperitoneal injection than after inhalation, while N-acetylcysteine was only effective after inhalation. The protective effects of mucolytics in the lung tissue may be due to their antioxidant activity together with other mechanisms.     

Speech and Swallowing in the Elderly

Normal speech and swallow depend on the integrity of the oral motor system and the underlying processes of respiration, phonation, sensation, resonance and articulation. A variety of age-related changes occur in the oral peripheral mechanism, some of which affect speech and swallow. Nonpathologic changes in the thoracic and laryngeal structures that serve to reduce the vital capacity of the lungs and produce perturbations in the acoustic signal alter the quality of the aging voice. As one ages, the oral mucosa reportedly thins, salivary flow may be decreased, and the sensory and motor integrity of the tongue musculature is altered. The effect of such changes on speech articulation and voice production in the elderly is discussed. Although not dependent on one another, speech and swallowing use many of the same oral structures and underlying physiologic mechanisms. The anatomic and physiologic changes in the aging pharvngeal area and oral tract that can affect swallowing are reviewd and studies of these changes are discussed. Viewing the oral area and vocal tract has required invasive, cumbersome or dangerous radiographic procedures, thereby limiting the quantity of research in this field. Recent work on the use of real-time ultrasound imaging to view the oral soft tissues and dynamic lingual gestures in vivo during speech and swallow are reviewed and ongoing studies of speech and swallowing performance in normal aging persons using this technique are presented.     

Influence of biosurfactants from probiotic bacteria on formation of biofilms on voice prostheses

Biofilms were grown on preconditioned voice prostheses with biosurfactants obtained from probiotic bacteria Lactococcus lactis 53 and Streptococcus thermophilus A in an artificial throat model. Both biosurfactants greatly reduced microbial numbers on prostheses and also induced a decrease in the airflow resistance that occurs on voice prostheses after biofilm formation. This study presents a promising strategy for prolonging the lifespan of voice prostheses.     

Influence of Biosurfactants from Probiotic Bacteria on Formation of Biofilms on Voice Prostheses

Biofilms were grown on preconditioned voice prostheses with biosurfactants obtained from probiotic bacteria Lactococcus lactis 53 and Streptococcus thermophilus A in an artificial throat model. Both biosurfactants greatly reduced microbial numbers on prostheses and also induced a decrease in the airflow resistance that occurs on voice prostheses after biofilm formation. This study presents a promising strategy for prolonging the lifespan of voice prostheses.     

The role of alginate in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell attachment

Among various functions, extracellular polymeric substances (EPS) provide microbial biofilms with mechanical stability and affect initial cell attachment, the first stage in the biofilm formation process. The role of alginate, an abundant polysaccharide in Pseudomonas aeruginosa biofilms, in the viscoelastic properties and adhesion kinetics of EPS was analyzed using a quartz crystal microbalance with dissipation (QCM-D) monitoring technology. EPS was extracted from two P. aeruginosa biofilms, a wild type strain, PAO1, and a mucoid strain, PAOmucA22 that over-expresses alginate production. The higher alginate content in the EPS originating from the mucoid biofilms was clearly shown to increase both the rate and the extent of attachment of the EPS, as well as the layer's thickness. Also, the presence of calcium and elevated ionic strength increased the thickness of the EPS layer. Dynamic light scattering (DLS) showed that the presence of calcium and elevated ionic strength induced intermolecular attractive interactions in the mucoid EPS molecules. For the wild type EPS, in the presence of calcium, an elevated shift in the distribution of the diffusion coefficients was observed with DLS due to a more compacted conformation of the EPS molecules. Moreover, the alginate over-expression effect on EPS adherence was compared to the effect of alginate over-expression on P. aeruginosa cell attachment. In a parallel plate flow cell, under similar hydraulic and aquatic conditions as those applied for the EPS adsorption tests in the QCM-D flow cell, reduced adherence of the mucoid strain was clearly observed compared to the wild type isogenic bacteria. The results suggest that alginate contributes to steric hindrance and shielding of cell surface features and adhesins that are known to promote cell attachment.     

Effects of chronic triclosan exposure upon the antimicrobial susceptibility of 40 ex-situ environmental and human isolates

BACKGROUND: Triclosan (TCS) exposure of Escherichia coli selects for tolerant clones, mutated in their enoyl-acyl carrier protein reductase (FabI). It has been inferred that this phenomenon is widespread amongst bacterial genera and might be associated with resistance to third party agents. METHODS: Ex-situ, low passage isolates of enteric, human axilla, human oral origin and bacteria isolated from a domestic drain, together with selected type cultures were exposed to escalating concentrations of TCS over 10 passages using a gradient plate technique. One fresh faecal isolate of E. coli was included as a positive control. TCS susceptibility was determined for all strains before and after exposure, whilst enteric isolates were additionally assessed for susceptibility towards chlorhexidine, tetracycline, chloramphenicol, nalidixic acid and ciprofloxacin, and the oral isolates towards chlorhexidine, tetracycline and metronidazole. RESULTS: Triclosan exposure of E. coli markedly decreased TCS susceptibility. TCS susceptibility also decreased for Klebsiella oxytoca, Aranicola proteolyticus and Stenotrophomonas maltophilia. Susceptibility of the remaining 35 strains to TCS and the other test agents remained unchanged. CONCLUSIONS: These data suggest that selection for high level resistance by TCS exposure is not widespread and appears to be confined to certain enteric bacteria, especially E. coli. Change in TCS susceptibility did not affect susceptibility towards chemically unrelated antimicrobials. SIGNIFICANCE AND IMPACT: Acquired high-level TCS resistance is not a widespread phenomenon.     

Effects of Quaternary Ammonium Silane Coatings on Mixed Fungal and Bacterial Biofilms on Tracheoesophageal Shunt Prostheses

Two quaternary ammonium silanes (QAS) were used to coat silicone rubber tracheoesophageal shunt prostheses, yielding a positively charged surface. One QAS coating [(trimethoxysilyl)-propyldimethyloctadecylammonium chloride] was applied through chemical bonding, while the other coating, Biocidal ZF, was sprayed onto the silicone rubber surface. The sprayed coating lost its stability within an hour, while the chemically bonded coating appeared stable. Upon incubation in an artificial throat model, allowing simultaneous adhesion and growth of yeast and bacteria, all coated prostheses showed significant reductions in the numbers of viable yeast (to 12% to 16%) and bacteria (to 27% to 36%) compared with those for silicone rubber controls, as confirmed using confocal laser scanning microscopy after live/dead staining of the biofilms. In situ hybridization with fluorescently labeled oligonucleotide probes showed that yeasts expressed hyphae on the untreated and Biocidal ZF-coated prostheses but not on the QAS-coated prostheses. Whether this is a result of the positive QAS coating or is due to the reduced number of bacteria is currently unknown. In summary, this is the first report on the inhibitory effects of positively charged coatings on the viability of yeasts and bacteria in mixed biofilms. Although the study initially aimed at reducing voice prosthetic biofilms, its relevance extends to all biomedical and environmental surfaces where mixed biofilms develop and present a problem.     

Effects of quaternary ammonium silane coatings on mixed fungal and bacterial biofilms on tracheoesophageal shunt prostheses

Two quaternary ammonium silanes (QAS) were used to coat silicone rubber tracheoesophageal shunt prostheses, yielding a positively charged surface. One QAS coating [(trimethoxysilyl)-propyldimethyloctadecylammonium chloride] was applied through chemical bonding, while the other coating, Biocidal ZF, was sprayed onto the silicone rubber surface. The sprayed coating lost its stability within an hour, while the chemically bonded coating appeared stable. Upon incubation in an artificial throat model, allowing simultaneous adhesion and growth of yeast and bacteria, all coated prostheses showed significant reductions in the numbers of viable yeast (to 12% to 16%) and bacteria (to 27% to 36%) compared with those for silicone rubber controls, as confirmed using confocal laser scanning microscopy after live/dead staining of the biofilms. In situ hybridization with fluorescently labeled oligonucleotide probes showed that yeasts expressed hyphae on the untreated and Biocidal ZF-coated prostheses but not on the QAS-coated prostheses. Whether this is a result of the positive QAS coating or is due to the reduced number of bacteria is currently unknown. In summary, this is the first report on the inhibitory effects of positively charged coatings on the viability of yeasts and bacteria in mixed biofilms. Although the study initially aimed at reducing voice prosthetic biofilms, its relevance extends to all biomedical and environmental surfaces where mixed biofilms develop and present a problem.     

Genomic identification of microbial species adhering to maxillofacial prostheses and susceptibility to different hygiene protocols

This study investigated the microbial colonization of maxillofacial prostheses and support tissues using the Checkerboard DNA–DNA hybridization method, and the efficacy of 0.12% chlorhexidine gluconate, 10% Ricinus communis solutions, or brushing, on colony forming unit (CFU) reduction in monospecies biofilms (Candida glabrata, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa) formed on two silicones (MDX 4-4210 and Bio-Skin). Biofilm was harvested from 43 maxillofacial prosthesis wearers for detection of 38 species of microorganisms. The CFU counts of the six above mentioned species were recorded after using the hygiene protocols. All 38 investigated species were identified in prostheses and tissues, with a higher prevalence in the prostheses. 0.12% chlorhexidine gluconate immersion showed the greatest antimicrobial effectiveness, followed by mechanical brushing protocols. MDX 4-4210 silicone produced lower CFU counts than Bio-Skin.     

A comparison of conventional SEM techniques, low temperature SEM and the electroscan wet scanning electron microscope to study the structure of a biofilm of Streptococcus crista CR3

Biofilms of Streptococcus crista CR3 were generated on hydroxyapatite (HA) discs for 20 h in a continuous flow system with brain heart infusion broth dripped over the disc at a rate of 6 ml h^-1. This study compares the conventional scanning electron microscope (SEM) preparation techniques, of critical point drying and freeze-drying, with low temperature SEM (LTSEM) and Electroscan generated images of hydrated biofilms, which preserve the integrity of hydrated polymers.
Critical point drying and freeze-drying caused almost complete disappearance of the matrix of extracellular polymeric substances (EPS). Critical point drying, however, showed evenly spaced single or paired cocci remaining on the HA disc whereas freeze-drying caused the biofilm to detach from the HA leaving only patchy clumps of cells visible. By comparison LTSEM preserved the EPS better than critical point drying and freeze-drying, but holes were seen in the top and side of the biofilm and the EPS did show some shrinkage artefacts. An untreated wet biofilm viewed in the Electroscan showed an intact, hydrated, smooth matrix of EPS with cell shapes only visible indistinctly in a canopy of moist EPS. No holes were visible and no shrinkage artefacts were evident. Therefore, Electroscan imaging of the biofilm was the only method that preserved the integrity of the matrix with no apparent shrinkage artefacts.     

Biofilms on tracheoesophageal voice prostheses: a confocal laser scanning microscopy demonstration of mixed bacterial and yeast biofilms

The aim of this study was to demonstrate the presence of yeast and bacterial biofilms on the surface of tracheoesophageal voice prostheses (TVPs) by a double-staining technique with confocal laser scanning microscopy (CLSM). Biofilms of 12 removed TVPs were visualized by scanning electron microscopy, then stained with ConA-FITC and propidium iodide for CLSM. Microbial identification was by partial 16S rRNA gene analysis and ITS-2 sequence analysis. Microbial biofilms on the TVPs consisted of bacteria and filamentous cells. Bacterial cells were attached to the filamentous and unicellular yeast cells, thus forming a network. Sequence analyses of six voice prostheses identified the presence of a variety of bacterial and yeast species. In vivo studies showed that Klebsiella oxytoca and Micrococcus luteus efficiently attached to Candida albicans. CLSM with double fluorescence staining can be used to demonstrate biofilm formations composed of a mixture of yeast and bacterial cells on the surface of TVPs.     

Voice rehabilitation of Broca's aphasia following total laryngectomy

Total laryngectomy, as a consequence of carcinoma of the larynx, results in loss of speech function. Cerebrovascular stroke is the leading cause of reduced speech production ability, and thereby communication difficulties. The case is presented of a 60-year-old male patient who suffered stroke five years after a total laryngectomy. Speech rehabilitation was hampered due to the depressive state of the patient. Although contraindicated, the secondary voice prosthesis was implanted. Only at that moment the patient showed willingness and motivation for speech rehabilitation. The aim of this presentation is to demonstrate that not all neurological disorders are contraindicated for implantation of voice prostheses.