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.     

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.     

Comparison of velocity profiles for different flow chamber designs used in studies of microbial adhesion to surfaces

Flow chambers are commonly used to study microbial adhesion to surfaces under environmentally relevant hydrodynamic conditions. The parallel plate flow chamber (PPFC) is the most common design, and mass transport occurs through slow convective diffusion. In this study, we analyzed four different PPFCs to determine whether the expected hydrodynamic conditions, which control both mass transport and detachment forces, are actually achieved. Furthermore, the different PPFCs were critically evaluated based on the size of the area where the velocity profile was established and constant with a range of flow rates, indicating that valid observations could be made. Velocity profiles in the different chambers were calculated by using a numerical simulation model based on the finite element method and were found to coincide with the profiles measured by particle image velocimetry. Environmentally relevant shear rates between 0 and 10,000 s(-1) could be measured over a sizeable proportion of the substratum surface for only two of the four PPFCs. Two models appeared to be flawed in the design of their inlets and outlets and allowed development of a stable velocity profile only for shear rates up to 0.5 and 500 s(-1). For these PPFCs the inlet and outlet were curved, and the modeled shear rates deviated from the calculated shear rates by up to 75%. We concluded that PPFCs used for studies of microbial adhesion to surfaces should be designed so that their inlets and outlets are in line with the flow channel. Alternatively, the channel length should be increased to allow a greater length for the establishment of the desired hydrodynamic conditions.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate.

The C function in Arabidopsis, which specifies stamen and carpel identity, is represented by a single gene called AGAMOUS (AG). From both petunia and cucumber, two MADS box genes have been isolated. Both share a high degree of amino acid sequence identity with the Arabidopsis AG protein. Their roles in specifying stamen and carpel identity have been studied by ectopic expression in petunia, resulting in plants with different floral phenotypes. Cucumber MADS box gene 1 (CUM1) induced severe homeotic transformations of sepals into carpelloid structures and petals into stamens, which is similar to ectopic AG expression in Arabidopsis plants. Overexpression of the other cucumber AG homolog, CUM10, resulted in plants with partial transformations of the petals into antheroid structures, indicating that CUM10 is also able to promote floral organ identity. From the two petunia AG homologs pMADS3 and Floral Binding Protein gene 6 (FBP6), only pMADS3 was able to induce homeotic transformations of sepals and petals. Ectopic expression of both pMADS3 and FBP6, as occurrs in the petunia homeotic mutant blind, phenocopies the pMADS3 single overexpresser plants, indicating that there is no additive effect of concerted expression. This study demonstrates that in petunia and cucumber, multiple AG homologs exist, although they differ in their ability to induce reproductive organ fate.     

A Quantitative and Dynamic Model of the Arabidopsis Flowering Time Gene Regulatory Network

Various environmental signals integrate into a network of floral regulatory genes leading to the final decision on when to flower. Although a wealth of qualitative knowledge is available on how flowering time genes regulate each other, only a few studies incorporated this knowledge into predictive models. Such models are invaluable as they enable to investigate how various types of inputs are combined to give a quantitative readout. To investigate the effect of gene expression disturbances on flowering time, we developed a dynamic model for the regulation of flowering time in Arabidopsis thaliana. Model parameters were estimated based on expression time-courses for relevant genes, and a consistent set of flowering times for plants of various genetic backgrounds. Validation was performed by predicting changes in expression level in mutant backgrounds and comparing these predictions with independent expression data, and by comparison of predicted and experimental flowering times for several double mutants. Remarkably, the model predicts that a disturbance in a particular gene has not necessarily the largest impact on directly connected genes. For example, the model predicts that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) mutation has a larger impact on APETALA1 (AP1), which is not directly regulated by SOC1, compared to its effect on LEAFY (LFY) which is under direct control of SOC1. This was confirmed by expression data. Another model prediction involves the importance of cooperativity in the regulation of APETALA1 (AP1) by LFY, a prediction supported by experimental evidence. Concluding, our model for flowering time gene regulation enables to address how different quantitative inputs are combined into one quantitative output, flowering time.     

Low-load compression testing: a novel way of measuring biofilm thickness

Biofilms are complex and dynamic communities of microorganisms that are studied in many fields due to their abundance and economic impact. Biofilm thickness is an important parameter in biofilm characterization. Current methods of measuring biofilm thicknesses have several limitations, including application, availability, and costs. Here, we present low-load compression testing (LLCT) as a new method for measuring biofilm thickness. With LLCT, biofilm thicknesses are measured during compression by inducing small loads, up to 5 Pa, corresponding to 0.1% deformation, making LLCT essentially a nondestructive technique. Comparison of the thicknesses of various bacterial and yeasts biofilms obtained by LLCT and by using confocal laser scanning microscopy (CLSM) resulted in the conclusion that CLSM underestimates the biofilm thickness due to poor penetration of different fluorescent dyes, especially through the thicker biofilms, whereas LLCT does not suffer from this thickness limitation.