Microbial biofilms on facial prostheses

The composition of microbial biofilms on silicone rubber facial prostheses was investigated and compared with the microbial flora on healthy and prosthesis-covered skin. Scanning electron microscopy showed the presence of mixed bacterial and yeast biofilms on and deterioration of the surface of the prostheses. Microbial culturing confirmed the presence of yeasts and bacteria. Microbial colonization was significantly increased on prosthesis-covered skin compared to healthy skin. Candida spp. were exclusively isolated from prosthesis-covered skin and from prostheses. Biofilms from prostheses showed the least diverse band-profile in denaturing gradient gel electrophoresis (DGGE) whereas prosthesis-covered skin showed the most diverse band-profile. Bacterial diversity exceeded yeast diversity in all samples. It is concluded that occlusion of the skin by prostheses creates a favorable niche for opportunistic pathogens such as Candida spp. and Staphylococcus aureus. Biofilms on healthy skin, skin underneath the prosthesis and on the prosthesis had a comparable composition, but the numbers present differed according to the microorganism.     

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.     

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.     

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.     

Candida biofilms on implanted biomaterials: a clinically significant problem

In recent years there has been an increasing appreciation that microbial biofilms are ubiquitous, which has resulted in a number of studies on infectious diseases from a biofilm perspective. Biofilms are defined as structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. A wide range of biomaterials used in clinical practice have been shown to support colonization and biofilm formation by Candida spp., and the increase in Candida infections in the last decades has almost paralleled the increase and widespread use of a broad range of medical implant devices, mainly in populations with impaired host defenses. Formation of Candida biofilms has important clinical repercussions because of their increased resistance to antifungal therapy and the ability of cells within biofilms to withstand host immune defenses. Further recognition and understanding of the role of Candida biofilms in human infection should help in the clinical management of these recalcitrant infections.     

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.     

Fatty acid analysis and spoilage potential of biofilms from two breweries

AIM: The microbial composition of biofilms from different locations of beer bottling plants were compared based on fatty acid profiles and correlated with the product-spoiling potential of these biofilms. METHODS AND RESULTS: The whole cell fatty acid profiles of 78 biofilms from bottling plants of two breweries were analysed. About half of the lipid profiles were dominated by oleic and linoleic acid, which refer to a high proportion of yeasts. In addition, more than half of all samples contained dimethylacetals indicating the presence of strictly anaerobic bacteria. Typical fatty acids for potentially beer-spoiling genera were detected in three biofilms. The majority of the biofilms contained no beer-spoiling organisms, as shown by inoculation experiments in beer. CONCLUSIONS: Biofilms from different locations of bottling plants were different with respect to their microbial composition. Potentially product-spoiling populations could be detected in a small number of samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilms on industrial plants can be characterized by a fast and cultivation-independent method with respect to overall microbial composition and presence of potentially product-spoiling micro-organisms.     

Microbial biodiversity in glacier-fed streams

While glaciers become increasingly recognised as a habitat for diverse and active microbial communities, effects of their climate change-induced retreat on the microbial ecology of glacier-fed streams remain elusive. Understanding the effect of climate change on microorganisms in these ecosystems is crucial given that microbial biofilms control numerous stream ecosystem processes with potential implications for downstream biodiversity and biogeochemistry. Here, using a space-for-time substitution approach across 26 Alpine glaciers, we show how microbial community composition and diversity, based on 454-pyrosequencing of the 16S rRNA gene, in biofilms of glacier-fed streams may change as glaciers recede. Variations in streamwater geochemistry correlated with biofilm community composition, even at the phylum level. The most dominant phyla detected in glacial habitats were Proteobacteria, Bacteroidetes, Actinobacteria and Cyanobacteria/chloroplasts. Microorganisms from ice had the lowest α diversity and contributed marginally to biofilm and streamwater community composition. Rather, streamwater apparently collected microorganisms from various glacial and non-glacial sources forming the upstream metacommunity, thereby achieving the highest α diversity. Biofilms in the glacier-fed streams had intermediate α diversity and species sorting by local environmental conditions likely shaped their community composition. α diversity of streamwater and biofilm communities decreased with elevation, possibly reflecting less diverse sources of microorganisms upstream in the catchment. In contrast, β diversity of biofilms decreased with increasing streamwater temperature, suggesting that glacier retreat may contribute to the homogenisation of microbial communities among glacier-fed streams.     

Examination of the genetic variability among biofilm-forming Candida albicans clinical isolates

Biofilms are microbial communities encased in a self-produced polymeric matrix and represent a common mode of microbial growth. Candida albicans is able to colonize the surface of catheters, prostheses, and epithelia, forming biofilms that are highly resistant to antimicrobial drugs. The objective of this study was the genotypic characterization of biofilm-forming C. albicans clinical isolates using RAPD (Random Amplified Polymorphic DNA). We have studied 25 clinical isolates of C. albicans from oral cavities, blood, skin, nail, stool, oesophagus biopsy and vaginal fluids from patients suffering from candidiasis. For each strain biofilm formation was analysed by measuring the ability to adhere to and grow on polystyrene plastic surfaces using XTT [2,3-bis(2-methoxi-4nitro-5sulfophenil)-2H tetrazolium-5carboxanilide] reduction assay. The similarity coefficients generated by RAPD using four different primers varied from 49 to 91%, indicating a high degree of genetic variability between the clinical isolates. The dendrogram clustered the isolates in four related groups, all groups included strains with very different abilities to form biofilms. The isolates with similar genotypes often showed very different biofilm formation abilities. Strains were grouped into clusters independently of their clinical sources. Our results suggested that a direct correlation does not exist between the biofilm-forming ability of natural populations of C. albicans and the genotype as determined by RAPD.     

Microflora on explanted silicone rubber voice prostheses: taxonomy, hydrophobicity and electrophoretic mobility

Silicone rubber voice prostheses are implants which are inserted in a non-sterile environment and therefore become quickly colonized by micro-organisms. The micro-organisms exist on the medical grade silicone rubber as mixed biofilms of bacteria and yeasts. A total of 79 bacterial and 39 yeast strains were isolated from these biofilms by soft ultrasonic treatment. Gram-positive/catalase-negative and Gram-positive/catalase-positive cocci represented the dominant bacterial strains. The yeasts were mainly Candida species. Further characterization of cell surface properties such as hydrophobicity by microbial adhesion to hexadecane and electrophoretic mobility showed a distinct difference when the bacterial strains were compared with the yeasts. The bacterial hydrophobicities ranged from 0 to 100% adhesion to hexadecane, whereas the yeast strains, especially the Candida albicans strains, all had markedly hydrophilic cell surfaces. A comparison of the electrophoretic mobilities showed also differences between bacteria and yeast. The values for the bacteria were found to be between -2.5 to -0.5 (10^-8 m² V^-1 s^-1), whereas for the yeasts electrophoretic mobilities were more positive. Based on the adhesive properties of the isolated micro-organisms, strategies can now be developed to modify the properties of the silicone rubber to reduce biofilm formation on such prostheses.     

Microbial community diversity and composition varies with habitat characteristics and biofilm function in macrophyte‐rich streams

Biofilms in streams play an integral role in ecosystem processes and function yet few studies have investigated the broad diversity of these complex prokaryotic and eukaryotic microbial communities. Physical habitat characteristics can affect the composition and abundance of microorganisms in these biofilms by creating microhabitats. Here we describe the prokaryotic and eukaryotic microbial diversity of biofilms in sand and macrophyte habitats (i.e. epipsammon and epiphyton, respectively) in five macrophyte‐rich streams in Jutland, Denmark. The macrophyte species varied in growth morphology, C:N stoichiometry, and preferred stream habitat, providing a range in environmental conditions for the epiphyton. Among all habitats and streams, the prokaryotic communities were dominated by common phyla, including Alphaproteobacteria, Bacteriodetes, and Gammaproteobacteria, while the eukaryotic communities were dominated by Stramenopiles (i.e. diatoms). For both the prokaryotes and eukaryotes, the epipsammon were consistently the most diverse communities and the epiphytic communities were generally similar among the four macrophyte species. However, the communities on the least complex macrophyte, Sparganium emersum, had the lowest richness and evenness and fewest unique OTUs, whereas the macrophyte with the most morphological complexity, Callitriche spp., had the highest number of unique OTUs. In general, the microbial taxa were ubiquitously distributed across the relatively homogeneous Danish landscape as determined by measuring the similarity among communities (i.e. Sørensen similarity index). Furthermore, we found significant correlations between microbial diversity (i.e. Chao1 rarefied richness and Pielou's evenness) and biofilm structure and function (i.e. C:N ratio and ammonium uptake efficiency, respectively); communities with higher richness and evenness had higher C:N ratios and lower uptake efficiency. In addition to describing the prokaryotic and eukaryotic community composition in stream biofilms, our study indicates that 1) physical habitat characteristics influence microbial diversity and 2) the variation in microbial diversity may dictate the structural and functional characteristics of stream biofilm communities.     

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.     

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 after 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.     

Living on the edge: biofilms developing in oscillating environmental conditions

Abstract

For the first time, the densities and diversity of microorganisms developed on ocean gliders were investigated using flow cytometry and Illumina MiSeq sequencing of 16S and 18S rRNA genes. Ocean gliders are autonomous buoyancy-driven underwater vehicles, equipped with sensors continuously recording physical, chemical, and biological parameters. Microbial biofilms were investigated on unprotected parts of the glider and surfaces coated with base, biocidal and chitosan paints. Biofilms on the glider were exposed to periodical oscillations of salinity, oxygen, temperature, pressure, depth and light, due to periodic ascending and descending of the vehicle. Among the unprotected surfaces, the highest microbial abundance was observed on the bottom of the glider’s body, while the lowest density was recorded on the glider’s nose. Antifouling paints had the lowest densities of microorganisms. Multidimensional analysis showed that the microbial communities formed on unprotected parts of the glider were significantly different from those on biocidal paint and in seawater.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.     

In vitro activity of amphotericin B and anidulafungin against Candida spp. biofilms

Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.     

Molecular characterization of subject-specific oral microflora during initial colonization of enamel

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.     

Characterization of the skin fungal microbiota in patients with atopic dermatitis and in healthy subjects

Patients with atopic dermatitis (AD) are highly susceptible to viral, bacterial, and fungal skin infections because their skin is dry and this compromises the barrier function of the skin. Therefore, the skin microbiota of patients with AD is believed to be different from that of healthy individuals. In the present study, the skin fungal microbiota of nine patients with mild, moderate, or severe AD and ten healthy subjects were compared using an rRNA clone library. Fungal D1/D2 large subunit analysis of 3647 clones identified 58 species and seven unknown phylotypes in face scale samples from patients with AD and healthy subjects. Malassezia species were predominant, accounting for 63%-86% of the clones identified from each subject. Overall, the non-Malassezia yeast microbiota of the patients was more diverse than that of the healthy individuals. In the AD samples 13.0 ± 3.0 species per case were detected, as compared to 8.0 ± 1.9 species per case in the samples taken from healthy individuals. Notably, Candida albicans, Cryptococcus diffluens, and Cryptococcus liquefaciens were detected in the samples from the patients with AD. Of the filamentous fungal microbiota, Cladosporium spp. and Toxicocladosporium irritans were the predominant species in these patients. Many pathogenic fungi, including Meyerozyma guilliermondii (anamorphic name, Candida guilliermondii), and Trichosporon asahii, and allergenic microorganisms such as Alternaria alternata and Aureobasidium pullulans were found on the skin of the healthy subjects. When the fungal microbiota of the samples from patients with mild/moderate to severe AD and healthy individuals were clustered together by principal coordinates analysis they were found to be clustered according to health status.     

Scanning electron microscopy of native biofilms on mung bean sprouts

Native biofilms present on the adaxial surface of cotyledons of mung bean sprouts (Vigna radiata) were studied by use of scanning electron microscopy. Biofilms were abundant on the cotyledon surfaces and were comprised of rod-shaped bacteria, cocci-shaped bacteria, or yeasts, often with one type of microbe predominant. In contrast to our earlier study of biofilms on green sprouts (alfalfa, clover, broccoli, and sunflower), yeast and cocci were abundant on mung bean. Filamentous fungi were not observed. Sheet-like or fibrillar material (presumably composed of secreted microbial polysaccharides, proteins, lipids, and nucleic acids) fully or partially covered the biofilms. Biofilms up to 5 mm in length were observed, and some biofilms were comprised of more than just a monolayer of microbial cells. Native biofilms on sprout surfaces undoubtedly play an important role in the ecology of plant epiphytic microbes and may also afford protected sites for plant and human bacterial pathogens.     

Microbial communities associated with skeletal tumors on Porites compressa

Coral tumors are atypical skeletal forms found on coral reefs worldwide. Here we present an analysis of the microbial communities associated with skeletal tumors on the coral Porites compressa. Microbial growth rates on both healthy and tumorous P. compressa were decoupled from the surrounding water column. Microbial communities associated with tumorous colonies had a significantly faster growth rate than those associated with healthy P. compressa. The microbial community associated with the tumors contained more culturable Vibrio spp. and could utilize more carbon sources than the microbes associated with healthy colonies. Presence of tumors affected the composition and dynamics of the microbial population associated with the entire colony.