Biofilm Metabolism Research Articles

Analyses of the Effects of Arginine, Nicotine, Serotype and Collagen-Binding Proteins on Biofilm Development by 33 Strains of Streptococcus mutans.

Streptococcus mutans serotype k strains comprise <3% of oral isolates of S. mutans but are prominent in diseased cardiovascular (CV) tissue. Collagen binding protein (CBP) genes, cbm and cnm, are prevalent in serotype k strains and are associated with endothelial cell invasion. Nicotine increases biofilm formation by serotype c strains of S. mutans, but its effects on serotype k strains and strains with CBP are unknown. Saliva contains arginine which alters certain properties of the extracellular polysaccharides (EPS) in S. mutans biofilm. We examined whether nicotine and arginine affect sucrose-induced biofilm of S. mutans serotypes k (n = 23) and c (n = 10) strains with and without CBP genes. Biofilm mass, metabolism, bacterial proliferation, and EPS production were assessed. Nicotine increased biomass and metabolic activity (p < 0.0001); arginine alone had no effect. The presence of a CBP gene (either cbm or cnm) had a significant effect on biofilm production, but serotype did not. Nicotine increased bacterial proliferation and the effect was greater in CBP + strains compared to strains lacking CBP genes. Addition of arginine with nicotine decreased both bacterial mass and EPS compared to biofilm grown in nicotine alone. EPS production was greater in cnm + than cbm + strains (p < 0.0001). Given the findings of S. mutans in diseased CV tissue, a nicotine induced increase in biofilm production by CBP + strains may be a key link between tobacco use and CV diseases.

Spatial alanine metabolism determines local growth dynamics of Escherichia coli colonies.

Bacteria commonly live in spatially structured biofilm assemblages, which are encased by an extracellular matrix. Metabolic activity of the cells inside biofilms causes gradients in local environmental conditions, which leads to the emergence of physiologically differentiated subpopulations. Information about the properties and spatial arrangement of such metabolic subpopulations, as well as their interaction strength and interaction length scales are lacking, even for model systems like Escherichia coli colony biofilms grown on agar-solidified media. Here, we use an unbiased approach, based on temporal and spatial transcriptome and metabolome data acquired during E. coli colony biofilm growth, to study the spatial organization of metabolism. We discovered that alanine displays a unique pattern among amino acids and that alanine metabolism is spatially and temporally heterogeneous. At the anoxic base of the colony, where carbon and nitrogen sources are abundant, cells secrete alanine via the transporter AlaE. In contrast, cells utilize alanine as a carbon and nitrogen source in the oxic nutrient-deprived region at the colony mid-height, via the enzymes DadA and DadX. This spatially structured alanine cross-feeding influences cellular viability and growth in the cross-feeding-dependent region, which shapes the overall colony morphology. More generally, our results on this precisely controllable biofilm model system demonstrate a remarkable spatiotemporal complexity of metabolism in biofilms. A better characterization of the spatiotemporal metabolic heterogeneities and dependencies is essential for understanding the physiology, architecture, and function of biofilms.

Efficacy of alpha-mangostin for antimicrobial activity against endodontopathogenic microorganisms in a multi-species bacterial-fungal biofilm model

ObjectiveTo determine the activity of alpha-mangostin on preformed bacterial-fungal multi-species biofilms in vitro, and to ascertain the impact on metabolic activity, biofilm structure and viability. DesignInhibitory concentrations (ICs) for alpha-mangostin against planktonic cultures of Candida albicans, Enterococcus faecalis, Lactobacillus rhamnosus, and Streptococcus gordonii were determined using a standard broth microdilution method. Single and multi-species (all species 1:1:1:1) biofilms were grown on polystyrene coverslips in Roswell Park Memorial Institute Medium for 48 h. The biofilms were then exposed to 0.2% (w/v) alpha-mangostin for 24 h. These concentrations were selected based on pilot experiments and the solubility of these compounds. 2% (v/v) chlorhexidine was used as a positive control and Roswell Park Memorial Institute Medium as a negative control. The metabolic activity of the biofilms after exposure was measured using metabolic (XTT) assays. Biofilms were visualised and quantified using fluorescent BacLight™ LIVE/DEAD staining. The biofilms were assessed for cell viability by culture and colony counting (CFU/mL). Results8 mg/L of alpha-mangostin was cidal against planktonic bacteria and 1000 mg/L for Candida. Alpha-mangostin was most active against L. rhamonosus biofilms and least active against C. albicans biofilm (metabolism inhibited by 99% and 78%, respectively). Alpha-mangostin exposure reduced the number of viable cells in the biofilms. ConclusionAlpha-mangostin inhibited the metabolic activity of bacterial-fungal biofilms effectively. The anti-biofilm activity of alpha-mangostin was comparable to chlorhexidine and thus has potential as a novel agent for endodontic therapy.

Design of Polymeric Thin Films to Direct Microbial Biofilm Growth, Virulence, and Metabolism.

Biofilms are ubiquitous in nature, yet strategies to direct biofilm behavior without genetic manipulation are limited. Due to the small selection of materials that have been used to successfully grow biofilms, the availability of functional materials that are able to support growth and program microbial functions remains a critical bottleneck in the design and deployment of functional yet safe microbes. Here, we report the design of insoluble pyridine-rich polymer surfaces synthesized using initiated chemical vapor deposition, which led to modulated biofilm growth and virulence in Pseudomonas aeruginosa (PAO1). A variety of extracellular virulence factors exhibited decreased production in response to the functional polymer, most significantly biomolecules also associated with iron acquisition, validating the material design strategy reported here. This report signifies a rich potential for materials-based strategies to direct the behavior of naturally occurring biofilms, which complement the existing genetic engineering toolkits in advancing microbiology, translational medicine, and biomanufacturing.

In vitro and in vivo analysis of monotherapy and dual therapy with ethyl caffeate and fluconazole on virulence factors of Candida albicans and systemic candidiasis

Candida albicans is the most clinically prevalent cause of systemic fungal infections in the immunocompromised population. The biofilm-forming ability of C. albicans confers resistance to conventional antifungal agents. The main aim of this study was to investigate the antifungal effects of ethyl caffeate (EC) alone and in combination with fluconazole (FLU) against C. albicans isolates. The single and combined antifungal activities of EC and FLU were evaluated against planktonic and biofilm cells of C. albicans by the checkerboard assay, time-kill test, crystal violet assay, live/dead staining, rhodamine 6G (R6G) efflux analysis and hydrolase activity. Monotherapy and dual therapy of EC and FLU against systemic candidiasis in a mouse model was also evaluated. The results showed that EC+FLU displayed synergism in 14/26 planktonic C. albicans isolates and 11/26 C. albicans biofilms with fractional inhibitory concentration index (FICI) values ranging between 0.06-0.49 and 0.02-0.38, respectively. Compared with monotherapy, the combination of EC+FLU can markedly inhibit adhesion, yeast-to-hyphae transition, premature and mature biofilm metabolism, hydrolase secretion and drug efflux function of C. albicans Z1407 and Z4935. Moreover, EC can potentiate the antifungal activity of FLU to improve mouse survival, reduce fungal burden and alleviate pathological damage in both C. albicans isolates compared with EC or FLU used alone. EC exhibits a moderate antifungal potential but can be a strong synergist with FLU against C. albicans, highlighting the potential of EC in clinical antifungal therapy as a sensitiser.

Altered Pseudomonas Strategies to Inhibit Surface Aspergillus Colonies.

Pseudomonas aeruginosa and Aspergillus fumigatus infections frequently co-localize in lungs of immunocompromised patients and individuals with cystic fibrosis (CF). The antifungal activity of P. aeruginosa has been described for its filtrates. Pyoverdine and pyocyanin are the principal antifungal P. aeruginosa molecules active against A. fumigatus biofilm metabolism present in iron-limited or iron-replete planktonic P. aeruginosa culture filtrates, respectively. Using various P. aeruginosa laboratory wild-type strains (PA14, PAO1, PAK), we found antifungal activity against Aspergillus colonies on agar. Comparing 36 PA14 and 7 PAO1 mutants, we found that mutants lacking both major siderophores, pyoverdine and pyochelin, display higher antifungal activity on agar than their wild types, while quorum sensing mutants lost antifungal activity. Addition of ferric iron, but not calcium or magnesium, reduced the antifungal effects of P. aeruginosa on agar, whereas iron-poor agar enhanced antifungal effects. Antifungal activity on agar was mediated by PQS and HHQ, via MvfR. Among the MvfR downstream factors, rhamnolipids and elastase were produced in larger quantities by pyoverdine–pyochelin double mutants and showed antifungal activity on agar. In summary, antifungal factors produced by P. aeruginosa on agar differ from those produced by bacteria grown in liquid cultures, are dependent on quorum sensing, and are downregulated by the availability of ferric iron. Rhamnolipids and elastase seem to be major mediators of Pseudomonas’ antifungal activity on a solid surface.

Transcriptional Changes in Bifidobacterium bifidum Involved in Synergistic Multispecies Biofilms.

Bifidobacterium bifidum is part of the core microbiota of healthy infant guts where it may form biofilms on epithelial cells, mucosa, and food particles in the gut lumen. Little is known about transcriptional changes in B. bifidum engaged in synergistic multispecies biofilms with ecologically relevant species of the human gut. Recently, we reported prevalence of synergism in mixed-species biofilms formed by the human gut microbiota. This study represents a comparative gene expression analysis of B. bifidum when grown in a single-species biofilm and in two multispecies biofilm consortia withBifidobacterium longum subsp. infantis, Bacteroides ovatus, and Parabacteroides distasonis in order to identify genes involved in this adaptive process in mixed biofilms and the influence on its metabolic and functional traits. Changes up to 58% and 43% in its genome were found when it grew in three- and four-species biofilm consortia, respectively. Upregulation of genes of B. bifidum involved in carbohydrate metabolism (particularly the galE gene), quorum sensing (luxS and pfs), and amino acid metabolism (especially branched chain amino acids) in both multispecies biofilms, compared to single-species biofilms, suggest that they may be contributing factors for the observed synergistic biofilm production when B. bifidum coexists with other species in a biofilm.

Comparison of the modulatory effects of three structurally similar potential prebiotic substrates on an in vitro multi-species oral biofilm

Previous research identified potential prebiotic substrates for oral health like the structural analogues N-acetyl-d-mannosamine (NADM) and N-acetyl-d-glucosamine (NADG). The main hypothesis of the current study was twofold. Firstly, it was hypothesized that the modulatory effects of NADM are not limited to changes in multi-species oral biofilm composition, but also include effects on metabolism, virulence, and inflammatory potential. Secondly, the presence and orientation of their N-acetyl group could play a role. Therefore, a comparison was made between the effects of NADM, NADG and d-(+)-mannose on multi-species oral biofilms. Besides a beneficial compositional shift, NADM-treated biofilms also showed an altered metabolism, a reduced virulence and a decreased inflammatory potential. At a substrate concentration of 1 M, these effects were pronounced for all biofilm aspects, whereas at ~ 0.05 M (1%(w/v)) only the effects on virulence were pronounced. When comparing between substrates, both the presence and orientation of the N-acetyl group played a role. However, this was generally only at 1 M and dependent on the biofilm aspect. Overall, NADM was found to have different effects at two concentrations that beneficially modulate in vitro multi-species oral biofilm composition, metabolism, virulence and inflammatory potential. The presence and orientation of the N-acetyl group influenced these effects.

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment.

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

Cytotoxicity and effects of curcumin and cinnamaldehyde hybrids on biofilms of oral pathogens

The objective of the study was to evaluate the cytotoxicity and effect of curcumin-cinnamaldehyde hybrids (CCHs) on the biofilm of oral pathogens. Of the 18 hybrids tested, nine had an inhibitory effect on at least one of the bacterial species tested, with minimal inhibitory and bactericidal concentrations ranging from 9 to 625 μg ml−1. CCH 7 promoted a potent inhibitory effect against all the bacterial species tested and better compatibility than chlorhexidine (CHX). CCH 7 also presented a similar or improved effect over that of CHX, causing a reduction in bacterial metabolism and viability in single and dual-species biofilms. CCH 7 reduced by 86% and 34% the viability of multispecies biofilms formed by collection and clinical strains. It can be concluded that CCH 7 was cytocompatible at the minimal inhibitory concentration, presented anti-biofilm action against oral pathogens, and could act as an antimicrobial agent for application in endodontics.

Assessment of Streptococcus mutans biofilms on orthodontic adhesives over 7 days

The purpose of this study was to compare the metabolism of Streptococcus mutans biofilms after 1-7days of growth on different orthodontic adhesives. Specimens of 6 commercial orthodontic adhesives were fabricated in custom-made molds and polymerized using a light-emitting diode light-curing unit. Bioluminescent S mutans (UA159:JM10) biofilms were grown on ultraviolet-sterilized specimens for 1, 3, 5, and 7days (n=18 biofilms/d/product) in anaerobic conditions at 37°C. The metabolism of biofilms (relative luminescence unit [RLU]) was measured 0, 2, 4, and 6minutes after exposure to D-luciferin solution using a microplate reader. A linear mixed-effects model was used to analyze the logarithm of RLU (log RLU). The model included fixed effects of products, days, and minutes. Tukey-Kramer post-hoc tests were then performed on the significant predictors of log RLU (α=0.05). Days (P<0.0001) and minutes (P<0.0001) were independent predictors of log RLU, but the products were not (P=0.5869). After adjusting for minutes, the log RLU was analyzed with a post-hoc test, and all differences between days were significant with the exceptions of day 3 from day 5 (P=0.0731) and day 5 from day 7 (P=0.8802). After adjusting for day, log RLU was analyzed with a post-hoc test and all differences in minutes were significant. No significant differences in the metabolism of S mutans biofilms were observed among the 6 orthodontic adhesives. Biofilms that were grown for 3days demonstrated the highest levels of biofilm metabolism as evidenced by higher mean log RLU values relative to 1, 5, and 7-day growth durations.

Salivary Pellicle Modification with Grape-seed Extract: In Vitro Study on the Effect on Bacterial Adhesion and Biofilm Formation.

Grape-seed extract (GSE) contains polyphenols that readily adhere to proteins and modify the acquired enamel pellicle (AEP). The first step in biofilm formation is bacterial adhesion to the AEP-covered enamel. The aim of this in vitro study was to test whether AEP modification with GSE, fluoride (F-), or their combination (GSE+F-) modulates bacterial adhesion, biofilm metabolism and composition, or cariogenic demineralisation of the enamel. The study comprised 3 parts: 1) single-strain Streptococcus gordonii species, 2) a five-species biofilm model, or 3) biofilm (re-)formation using the five-species biofilm model after removal of initial biofilm with toothbrushing. Human whole-mouth stimulated saliva was used to form an AEP on human enamel specimens. The AEP was incubated in water (control), or modified with GSE, F-, or GSE+F-. Bacterial adhesion, biofilm diversity, metabolic activity, biofilm mass, and cariogenic demineralisation (surface hardness) of enamel were assessed after incubation in bacterial broths after 4 h or 22 h. Differences between groups were analysed with one-way ANOVA and post-hoc Bonferroni tests. GSE and GSE+F- statistically significantly decreased single-strain S. gordonii adhesion, but had no relevant influence when the five-species biofilm model was used. In the biofilm (re-)formation model, GSE reduced bacterial adhesion compared to GSE+F-, while F- caused less cariogenic demineralisation than was found in the control group. AEP modified with GSE retards S. gordonii adhesion, but it does not influence the formation, metabolism and composition of a cariogenic multi-species biofilm.

Molecular Modifications of the Pseudomonas Quinolone Signal in the Intermicrobial Competition with Aspergillus.

The Pseudomonas quinolone signal (PQS) is an important quorum-sensing molecule for Pseudomonas aeruginosa that regulates virulence factors, chelates iron, and is an important factor in interactions with eukaryotes, including fungi and mammalian hosts. It was previously shown to inhibit or boost Aspergillus, depending on the milieu iron concentration. We studied several molecular modifications of the PQS molecule, and their effects on Aspergillus biofilm metabolism and growth in vitro, and the effects of iron supplementation. We found that most molecules inhibited Aspergillus at concentrations similar to that of PQS, but with relatively flat dose-responses, and all were less potent than PQS. The inhibition was reversible by iron, suggesting interference with fungal iron metabolism. Stimulation of Aspergillus was not noted. We conclude that the critical Aspergillus-inhibiting moeities of the PQS molecule were partially, but not completely, interfered with by molecular modifications at several sites on the PQS molecule. The mechanism, as with PQS, appears to relate to fungal iron metabolism.

Pseudomonas aeruginosa Virulence Factors Support Voriconazole Effects on Aspergillus fumigatus

Pseudomonas aeruginosa and Aspergillus fumigatus are pathogens that are associated with deterioration of lung function, e.g., in persons with cystic fibrosis (CF). There is evidence that co-infections with these pathogens cause airway inflammation and aggravate pathology in CF lungs. Intermicrobial competition of P. aeruginosa and A. fumigatus has been described, but it is unknown how anti-fungal therapy is affected. The anti-fungal azole voriconazole (VCZ), supernatants of P. aeruginosa laboratory isolates PA14 or PAO1, or clinical isolate Pa10 independently inhibited biofilm metabolism of A. fumigatus isolates 10AF and AF13073. When VCZ and supernatants were combined at their IC50s, synergistic effects on A. fumigatus were found. Synergistic effects were no longer observed when P. aeruginosa supernatants were prepared in the presence of iron, or when P. aeruginosa mutants were lacking the ability to produce pyoverdine and pyochelin. Combination of pure P. aeruginosa products pyoverdine, pyochelin, and pyocyanin with VCZ showed synergistic anti-fungal effects. Combining VCZ with P. aeruginosa supernatants also improved its MIC and MFC against planktonic A. fumigatus. In summary, in the case of P. aeruginosa–A. fumigatus co-infections, it appeared that the P. aeruginosa co-infection facilitated therapy of the Aspergillus; lower concentrations of VCZ might be sufficient to control fungal growth.

Inhibition of Candida albicans and Mixed Salivary Bacterial Biofilms on Antimicrobial Loaded Phosphated Poly(methyl methacrylate).

Biofilms play a crucial role in the development of Candida-associated denture stomatitis. Inhibition of microbial adhesion to poly(methyl methacrylate) (PMMA) and phosphate containing PMMA has been examined in this work. C. albicans and mixed salivary microbial biofilms were compared on naked and salivary pre-conditioned PMMA surfaces in the presence or absence of antimicrobials (Cetylpyridinium chloride [CPC], KSL-W, Histatin 5 [His 5]). Polymers with varying amounts of phosphate (0–25%) were tested using four C. albicans oral isolates as well as mixed salivary bacteria and 24 h biofilms were assessed for metabolic activity and confirmed using Live/Dead staining and confocal microscopy. Biofilm metabolism was reduced as phosphate density increased (15%: p = 0.004; 25%: p = 0.001). Loading of CPC on 15% phosphated disks showed a substantial decrease (p = 0.001) in biofilm metabolism in the presence or absence of a salivary pellicle. Salivary pellicle on uncharged PMMA enhanced the antimicrobial activity of CPC only. CPC also demonstrated remarkable antimicrobial activity on mixed salivary bacterial biofilms under different conditions displaying the potent efficacy of CPC (350 µg/mL) when combined with an artificial protein pellicle (Biotene half strength).

A Novel Small Molecule, LCG-N25, Inhibits Oral Streptococcal Biofilm.

Dental caries is a chronic oral infectious disease caused by cariogenic biofilm adhered on the tooth surface. Our previous study demonstrated that a repurposed natural compound napabucasin (NAP) showed good antimicrobial activity against oral streptococcal biofilms. The current study designed a novel small molecule, namely LCG-N25, using NAP as a lead compound, and aimed to investigate its potential as an antimicrobial agent in the control of dental caries. LCG-N25 was designed and synthesized with reference to the structure of NAP. The minimal inhibitory concentrations and the minimal bactericidal concentrations of LCG-N25 against Streptococcus mutans, Streptococcus sanguinis, and Streptococcus gordonii were evaluated by microdilution method. The antimicrobial activity of LCG-N25 was further evaluated by crystal violet staining, colony forming units counting, biofilm metabolism assay, dead/live fluorescent staining, and scanning electron microscopy. The effect of LCG-N25 on the extracellular polysaccharides of biofilms was determined by both anthrone-sulfuric acid method and fluorescent staining. The microbial composition of streptococcal biofilms after LCG-N25 treatment was further visualized and quantified by fluorescence in situ hybridization. Besides, the cytotoxicity of LCG-N25 was evaluated by Cell Counting Kit-8 assay, and repeated exposure of S. mutans to LCG-N25 treatment was performed to assess if this novel compound could induce drug resistance of this cariogenic bacterium. We found that LCG-N25 exhibited a good antibacterial activity, low-cytotoxicity, and did not induce drug resistance of cariogenic S. mutans. These findings suggest that LCG-N25 may represent a promising antimicrobial agent that can be used as an adjuvant to the management of dental caries.

Cinnamaldehyde and α-terpineol as an alternative for using as denture cleansers: antifungal activity and acrylic resin color stability

Objective: here was investigated the potential of phytoconstituents as cinnamaldehyde and α-terpineol as denture cleansers, which was evaluated the antifungal activity, also their effect on the color change of the poly(methyl)methacrylate (PMMA). Methodology: Candida albicans biofilms were formed on PMMA specimens and exposed a 10 minutes daily treatment with cinnamaldehyde (10 mg/ mL), α-terpineol (10 mg/ mL), NaCl 0.9%, and NaClO 1%. Biofilms were collected, then analyzed the cell viability (CFU/ mL), cell metabolism (MTT), and roughness. Moreover, the color variation of the specimens during seven and 14 days was checked. Kruskal-Wallis, Mann Whitney, and ANOVA one-way were applied (α &lt; 0.05). Results: cinnamaldehyde and α-terpineol decrease the number of viable cells compared to NaCl 0.9% by 76% and 83.2%, respectively (p&lt; 0.05). Both substances reduce biofilm metabolism by around 70% (p&lt; 0.05). Also, the phytoconstituents showed low biofilm roughness, similar to NaClO (p&gt; 0.05), without changing the color of the acrylic resin (p&gt; 0.05). Conclusion: cinnamaldehyde and α-terpineol, at a concentration of 10 mg/ mL, shows antifungal activity on C. albicans biofilms free from PMMA surface changes. From a clinical viewpoint, these substances have a strong potential to be incorporated into denture cleansers solutions.

Association of different Candida species with catheter-related candidemia, and the potential antifungal treatments against their adhesion properties and biofilm-forming capabilities.

BackgroundTo compare the adhesion properties and biofilm‐forming capabilities of 27 Candida isolates obtained from catheter‐related candidemia patients and to evaluate the inhibitory effects of antifungal agents on different Candida species.Material and MethodsSeven C. albicans, six C. parapsilosis, five C. guilliermondii, five C. tropicalis, and four C. glabrata clinical isolates were investigated. We quantified the adherence of these Candida species by flow cytometric method and evaluated the formation of biofilms by XTT reduction and crystal violet methods. Actions of micafungin (MF), fluconazole (FZ), and N‐acetylcysteine (NAC) on the adhesion and biofilm formation of different Candida species were determined.ResultsNon‐albicans Candida species were demonstrated to have stronger adhesion abilities compared with C. albicans. The biofilm‐forming capabilities of different Candida species were varied considerably, and the degree of biofilm formation might be affected by different assay approaches. Interestingly, C. parapsilosis displayed the highest biofilm formation abilities, while C. glabrata exhibited the lowest total biomass and metabolic activity. Furthermore, the inhibitory activities of MF, FZ, and NAC on fungal adhesion and biofilm formation were evaluated, and the results indicated that MF could reduce the adhesion ability and biofilm metabolism more significantly (p < 0.05), and its antifungal activity was elevated in a dose‐dependent manner.ConclusionNon‐albicans Candida species, especially C. guilliermondii, C. tropicalis, and C. parapsilosis, exhibited higher adhesion ability in catheter‐related candidemia patients. However, these Candida species had varied biofilm‐forming capabilities. MF tended to have stronger inhibitory effects against both adhesion and biofilm formation of different Candida species.

Interactive effects of discharge reduction and fine sediments on stream biofilm metabolism.

Discharge reduction, as caused by water diversion for hydropower, and fine sediments deposition, are prevalent stressors that may affect multiple ecosystem functions in streams. Periphytic biofilms play a key role in stream ecosystem functioning and are potentially affected by these stressors and their interaction. We experimentally assessed the interactive effects of discharge and fine sediments on biofilm metabolism in artificial indoor channels using a factorial split-plot design with two explanatory variables: water discharge (20, 39, 62, 141 and 174 cm3 s-1) and fine sediments (no sediment or 1100 mg L-1 of sediments). We incubated artificial tiles for 25 days in an unpolluted stream to allow biofilm colonization, and then placed them into the indoor channels for acclimation for 18 days. Subsequently, we manipulated water discharge and fine sediments and, after 17 days, we measured biofilm chlorophyll-a concentration and metabolism. Water velocity (range, 0.5 to 3.0 cm s-1) and sediment deposition (range, 6.1 to 16.6 mg cm-2) increased with discharge, the latter showing that the effect of increased inputs prevailed over sloughing. In the no-sediment treatments, discharge did not affect biofilm metabolism, but reduced chlorophyll-a. Sediments, probably as a consequence of nutrients released, promoted metabolism of biofilm and chlorophyll-a, which became independent of water discharge. Our results indicate that pulses of fine sediments can promote biofilm algal biomass and metabolism, but show interactive effects with discharge. Although discharge reduction can affect the abundance of basal resources for food webs, its complex interactions with fine sediments make it difficult to forecast the extent and direction of the changes.

Ratiometric imaging of extracellular pH in Streptococcus mutans biofilms exposed to different flow velocities and saliva film thicknesses

ABSTRACT Introduction: Fluid flow has a prominent influence on the metabolism of surface-attached biofilms. Dental biofilms are covered by a thin saliva film that flows at different rates in different locations under stimulated and unstimulated conditions. Methods:The present study employed pH ratiometry to study the impact of different flow velocities, saliva film thicknesses and saliva concentrations on microscale pH developments in Streptococcus mutans biofilms of different age. Results:While saliva flow at a velocity of 0.8 mm/min (unstimulated flow) had little impact on biofilm pH, stimulated flow (8 mm/min; 80 mm/min) affected vertical pH gradients in the biofilms and raised the average pH in 48-h biofilms, but not in 72-h and 168-h biofilms. The saliva film thickness had a strong impact on biofilm pH under both static and dynamic conditions. pH drops were significantly higher in biofilms exposed to a thin saliva film (≤ 50 µm) than a thick saliva film (> 50 µm). pH drops in the biofilms were also strongly dependent on the saliva concentration and thus the buffer capacity of the salivary medium. For 48-h and 72-h biofilms, but not for 168-h biofilms, pH drops in distinct microenvironments were more pronounced when the local biofilm thickness was high.