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.     

Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides. The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25–100 μg ml⁻¹. To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilisBE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 μg ml⁻¹ decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures. B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed. This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion. Received 27 October 1998/ Accepted in revised form 21 February 1999     

Selective antimicrobial activity of chitosan on beer spoilage bacteria and brewing yeasts

Chitosan (0.1 g l(-1)), assayed in a simple medium, reduced the viability of four lactic acid bacteria isolated during the beer production process by 5 logarithmic cycles, whereas activity against seven commercial brewing yeasts required up to 1 g chitosan l(-1). Antimicrobial activity was inversely affected by the pH of the assay medium. In brewery wort, chitosan (0.1 g l(-1)) selectively inhibited bacterial growth without altering yeast viability or fermenting performance.     

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.     

The influence of the leaflets' curvature on the flow field in two bileaflet prosthetic heart valves

A successful mechanical prosthetic heart valve design is the bileaflet valve, which has been implanted for the first time more than 20 years ago. A key feature of bileaflet valves is the geometry of the two leaflets, which can be very important in determining the flow field. Laser Doppler anemometry (LDA) was used to perform an accurate study of the velocity and turbulence shear stress peak values (TSS(max)) fields at four distances from the valve plane. TSS(max) is a relevant parameter to assess the risk of hemolysis and platelet activation associated to the implantation of a prosthetic device, continuously interacting with blood. Two bileaflet valves were tested: the St. Jude HP and the Sorin Bicarbon, of the same nominal size (19mm). The former has flat leaflets, whereas the latter's leaflets have a cylindrical surface. A high regime (CO: 6l/min) was imposed, in order to test the two valves at maximum Reynolds number and consequent turbulence generation. The flat-leaflet design of the St. Jude generates a TSS field constant with distance; on the contrary, the Bicarbon's shear stress field undergoes an evident development, with an unexpected central peak at a distance comparable to the valve's dimensions (21mm). The two bileaflet valves tested, although very similar in design, behave very differently as for their turbulence properties. In particular, the concept of curved wake leads to conclude that the curvature of the leaflets' surface must be identified as an important parameter, which deserves careful attention in PHV design and development.     

Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi

Three antimicrobial peptides exhibiting in vitro antifungal activity were expressed in Arabidopsis to compare their in planta activity. β-Purothionin, cecropin B, and phor21 were expressed under an endogenous promoter with moderate-level activity and excreted extracellularly. Expression of β-purothionin rendered the greatest antibacterial and antifungal resistance while cecropin B enhanced only antibacterial activity and phor21 did not improve antimicrobial resistance. The transgenic β-purothionin arrested fungal growth on leaf surfaces and infection of stomata. Leaf extracts from plants producing β-purothionin and cecropin B displayed membrane permeabilizing activity. The in planta antimicrobial activity of the tested peptides was consistent with previously reported in vitro experiments. The expression strategy allowed enhanced antifungal resistance without high-level transgene expression.Electronic Supplementary Material Supplementary material is available for this article at     

The structure and matrix dynamics of bacterial biofilms as revealed by antimicrobial peptides' diffusion

From the biological point of view, bacterial biofilms are communities of bacteria embedded in a self-produced gel matrix composed of polysaccharides, DNA, and proteins. Considering the biophysical point of view, the biofilm matrix is a highly dense, crowded medium that imposes constraints to solute diffusion, depending on the size, conformational dynamics, and net charge. From the pharmacological point of view, biofilms are additional difficulties to drug development as heterogeneity in oxygen and nutrient distribution, and consequently, heterogeneity in bacterial metabolic status leads to recalcitrance. For peptide scientists, biofilms are both a challenge and an opportunity. Biofilms can be intruded by peptides, revealing important biological, biophysical, and pharmacological insights. Peptides can be engineered for different sizes, flexibilities, and net charges, unravelling the determinants of diffusion; they kill bacteria by lysis, overcoming the hurdles of metabolic status heterogeneity, and they are able to kill bacteria in the biofilm core, leaving the matrix intact, that is, without causing bacterial biofilm dispersion as side effect. This concise review addresses the knowledge reached while interrogating bacterial biofilms with peptides and other reporter molecules, and the advances therefrom in biology, biophysics, and drug development.     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the “carpet-model” and thus, provide important insights into their antimicrobial mechanisms.     

Atomic force microscopy study of the antimicrobial action of Sushi peptides on Gram negative bacteria

The antibacterial effect of the endotoxin-binding Sushi peptides against Gram-negative bacteria (GNB) is investigated in this study. Similar characteristics observed for Atomic force microscopy (AFM) images of peptide-treated Escherichia coli and Pseudomonas aeruginosa suggest that the Sushi peptides (S3) evoke comparable mechanism of action against different strains of GNB. The results also indicate that the Sushi peptides appear to act in three stages: damage of the bacterial outer membrane, permeabilization of the inner membrane and disintegration of both membranes. The AFM approach has provided vivid and detailed close-up images of the GNB undergoing various stages of antimicrobial peptide actions at the nanometer scale. The AFM results support our hypothesis that the S3 peptide perturbs the GNB membrane via the "carpet-model" and thus, provide important insights into their antimicrobial mechanisms.     

Long-run monitoring of bacteria,yeasts and other micromycetes in the air of an industrial conurbation

Three different methods were used for the monitoring of airborne microorganisms: (1). Cultivation of microbes trapped in a single-stage biological impactor directly on a solid agar nutrient medium (meat-pepton agar, Sabouraud's agar, blood agar) in Petri dishes. The repeated yearly course of concentrations of cultivable organisms, or colony-forming units (CFU), was obtained by long-run measurements. (2) Aeresol was trapped by impact on membrane filters, and the microorganisms were cultivated by placing the filters on the agar media as above. (3) Direct microorganism counting in a fluorescence microscope; air was sampled in a four-stage impactor where the aerosol was trapped on microscope slides, and the microorganisms were subsequently stained with fluorescent dyes (fluorescein diacctate, 4;6-diamidino-2-phenylindole and, particular, ethidium bromide).

The highest microorganism counts were obtained by using the fluorescence method, the direct cultivation method gave counts an order of magnitude lower, and the method of cultivation on filters gave values approximately 10 times lower than the conventional cultivation.

High variations in the airborne CFU concentrations over the year were observed in Prague. Over the winter season the variations in the amounts of airborne bacteria and other micromycetes as well as the amounts themselves were lower than in the remaining seasons. In the spring and in the summer, the concentrations of yeasts and other micromycetes were highest, whereas in the autumn the concentrations of the microorganisms decreased. Among the bacteria cultivated form the airborne aerosol, the genera Micrococcus, Bacillus, Neisseria and Corynebacterium predominated. The prevailing genera of micromycetes were Penicillium, Aspergillus and Cladosporium.

The concentrations of microorganisms in free air were also affected by the local weather conditions, temperature in particular, the overall air pollution by aerosols was of minor importance in this respect.     

The influence of chloroquine on the formation of porphyrins by yeasts

A single two-compartment model suitable for studying the production and elimination of porphyrins from cells was prepared. Chloroquine with increasing concentrations:-

• 1.1. Inhibits the total production of porphyrins.
• 2.2. Reduces intracellular concentration of porphyrins.
• 3.3. Increases transversal permeation of porphyrins through the cellular membrane.

     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

Novel aqueous oil‐in‐water emulsions containing extracts of natural coniferous resins are strongly antimicrobial against enterobacteria,staphylococci and yeasts,as well as on bacterial biofilms

Aims

The aim of this study was to examine the antimicrobial properties of novel aqueous natural rapeseed oil/saline emulsions containing different soluble components of spruce resin.

Methods and Results

The composition of aqueous resin emulsions was analysed by GC‐MS and their antimicrobial properties were studied with challenge tests and with turbidometric assays. The emulsions were strongly antimicrobial against common Gram‐positive and Gram‐negative bacteria (including MRSA) as well as common yeasts. Furthermore, they inhibited the biofilm formation and eradicated the microbial biofilms on tested microbes. Characteristic for the emulsions was the presence of oxidized resin acids. Other main components present in emulsions, such as lignans and coumaric acids, were not antimicrobial, when tested separately.

Conclusions

The results indicated that the oxidized resin acids were the antimicrobial components in the emulsions. Also, there appears to be a stoichiometric relationship between the number of resin acid molecules and the number microbe cells in the antimicrobial action.

Significance and Impact of the Study

The fact that these solutions do not contain abietic acid, which is the main allergenic compound in resins, suggests that these solutions would be suitable, well‐tolerated antimicrobials for various medical applications. The aqueous formulation will also allow the expansion of the use of these emulsions in from medical applications to the food preservatives and disinfectants.     

气体环境对厌氧益生菌乳双歧杆菌V9菌株生长影响的研究

<正>乳双歧杆菌(Bifidobacterium lactis)V9菌株分离自健康蒙古族儿童肠道,已被广泛应用于开发各类益生菌产品[1]。由于双歧杆菌为厌氧菌[2],因此培养和保存时的气体环境会影响其活菌数量和益生功效,也成为该菌实现产业化道路上必须关注的重要环节。本刊于2012年第7期刊登了其木格苏都、张和平等的文章"不同气体环境对益生菌Bifidobacterium lactis V9生长的影响"[3]。作者对该菌在不同气体环境中的生长代谢特性进行了系统研究,明确了在有微量二氧化碳和氧气存在的环境下更有利于其生长。该研究为提高乳双歧