Mixed-species Biofilm Formation Research Articles

A novel multiplex fluorescent-labeling method for the visualization of mixed-species biofilms in vitro.

In nature, bacteria usually exist as mixed-species biofilms, where they engage in a range of synergistic and antagonistic interactions that increase their resistance to environmental challenges. Biofilms are a major cause of persistent infections, and dispersal from initial foci can cause new infections at distal sites thus warranting further investigation. Studies of development and spatial interactions in mixed-species biofilms can be challenging due to difficulties in identifying the different bacterial species in situ. Here, we apply CellTrace dyes to studies of biofilm bacteria and present a novel application for multiplex labeling, allowing identification of different bacteria in mixed-species, in vitro biofilm models. Oral bacteria labeled with CellTrace dyes (far red, yellow, violet, and CFSE [green]) were used to create single- and mixed-species biofilms, which were analyzed with confocal spinning disk microscopy (CSDM). Biofilm supernatants were studied with flow cytometry (FC). Both Gram-positive and Gram-negative bacteria were well labeled and CSDM revealed biofilms with clear morphology and stable staining for up to 4 days. Analysis of CellTrace labeled cells in supernatants using FC showed differences in the biofilm dispersal between bacterial species. Multiplexing with different colored dyes allowed visualization of spatial relationships between bacteria in mixed-species biofilms and relative coverage by the different species was revealed through segmentation of the CSDM images. This novel application, thus, offers a powerful tool for studying structure and composition of mixed-species biofilms in vitro.IMPORTANCEAlthough most chronic infections are caused by mixed-species biofilms, much of our knowledge still comes from planktonic cultures of single bacterial species. Studies of formation and development of mixed-species biofilms are, therefore, required. This work describes a method applicable to labeling of bacteria for in vitro studies of biofilm structure and dispersal. Critically, labeled bacteria can be multiplexed for identification of different species in mixed-species biofilms using confocal spinning disk microscopy, facilitating investigation of biofilm development and spatial interactions under different environmental conditions. The study is an important step in increasing the tools available for such complex and challenging studies.

Multiomics reveals the mechanism of B. longum in promoting the formation of mixed-species biofilms.

It has been found previously that Bifidobacterium longum, Bacteroides ovatus, Enterococcus faecalis, and Lactobacillus gasseri can form a biofilm better when co-cultured in vitro and B. longum is the core biofilm-formation-promoting strain in this community. B. longum is part of the core microbiota in the gut and is widely recognized as a probiotic. Therefore, it is necessary to explore its role in mixed-species biofilms through transcriptomics and metabolomics. Metabolomics showed that the increase in amino acid and purine content could promote biofilm formation. In transcriptomic analysis, many genes related to carbohydrate metabolism, amino acid metabolism, and environmental tolerance of B. longum were up-regulated. Combined with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis, the differentially expressed genes (DEGs) of B. longum in mixed-species biofilms were mainly correlated to "quorum sensing (QS)", "ABC transporters", "biosynthesis of amino acids", "microbial metabolism in different environments", "carbohydrate metabolism" and "two-component system". In addition, the rpl and rps gene families, which function in the metabolism of organic substances and the biosynthesis of amino acids, were the core DEGs according to the analysis of the protein-protein interaction (PPI) network. Finally, by combining metabolomics and quorum sensing mechanisms, it was found that the metabolism of autoinducer peptides (proliylglycine and glycylleucine), N-acyl homoserine lactone (N-(3-oxo hydroxy) homoserine lactone), and AI-2 can promote the formation of biofilms, both mono- and mixed-species biofilms composed of B. longum. Our research enabled us to understand the critical role of B. longum in mixed-species biofilms and the interactions between biofilm metabolism and gut health. In addition, the generated knowledge will be of great significance for us to develop biofilm products with beneficial functions in future.

Characterization of Mixed-Species Biofilms Formed by Four Gut Microbiota.

In natural settings, approximately 40-80% of bacteria exist as biofilms, most of which are mixed-species biofilms. Previous studies have typically focused on single- or dual-species biofilms. To expand the field of study on gut biofilms, we found a group of gut microbiota that can form biofilms well in vitro: Bifidobacterium longum subsp. infantis, Enterococcus faecalis, Bacteroides ovatus, and Lactobacillus gasseri. The increase in biomass and bio-volume of the mixed-species biofilm was confirmed via crystal violet staining, field emission scanning electron microscopy, and confocal laser scanning microscopy, revealing a strong synergistic relationship in these communities, with B. longum being the key biofilm-contributing species. This interaction may be related to changes in the cell number, biofilm-related genes, and metabolic activities. After quantifying the cell number using quantitative polymerase chain reaction, B. longum and L. gasseri were found to be the dominant flora in the mixed-species biofilm. In addition, this study analyzed biological properties of mixed-species biofilms, such as antibiotic resistance, cell metabolic activity, and concentration of water-insoluble polysaccharides. Compared with single-species biofilms, mixed-species biofilms had higher metabolic activity, more extracellular matrix, and greater antibiotic resistance. From these results, we can see that the formation of biofilms is a self-protection mechanism of gut microbiota, and the formation of mixed-species biofilms can greatly improve the survival rate of different strains. Finally, this study is a preliminary exploration of the biological characteristics of gut biofilms, and the molecular mechanisms underlying the formation of biofilms warrant further research.

Streptococcus mutans and Candida albicans Biofilm Inhibitors Produced by Lactiplantibacillus plantarum CCFM8724.

Lactiplantibacillus plantarum CCFM8724 inhibits the growth of Streptococcus mutans and Candida albicans in mixed-species biofilm formation. In this study, bioactive compound including cyclo (leu-pro), cyclo (phe-pro), and some organic acids, such as 3-phenyllactic acid, hydrocinnamic acid, and palmitic acid, were identified through GC-MS analysis. At 50μg·mL-1, cyclo (leu-pro) reduced biofilm mass (OD600) from 3.00 to 2.00, and hydrocinnamic acid at 25μg·mL-1 reduced biofilm mass (OD600) from 3.00 to 1.00. The expression of ALS3 and HWP1 was downregulated by cyclo (leu-pro). Furthermore, a mixture of cyclo (leu-pro), cyclo (phe-pro), 3-phenyllactic acid, hydrocinnamic acid, and palmitic acid, had anti-biofilm activity. Overall, the results provide promising baseline information for the potential use of this probiotic and its components in preventing biofilm formation.

Effects of lipid emulsions on the formation of Escherichia coli\u2013Candida albicans mixed-species biofilms on PVC

Patients receiving lipid emulsions are at increased risk of contracting catheter-related bloodstream infections (CRBSIs) in the clinic. More than 15% of CRBSIs are polymicrobial. The objective of this study was to explore the effects of lipid emulsions on the formation of Escherichia coli (E. coli)–Candida albicans (C. albicans) mixed-species biofilms (BFs) on polyvinyl chloride (PVC) surfaces and the underlying mechanism. Mixed-species BFs were produced by coculturing E. coli and C. albicans with PVC in various concentrations of lipid emulsions. Crystal violet staining and XTT assays were performed to test the mixed-species BF biomass and the viability of microbes in the BFs. The microstructures of the BFs were observed by an approach that combined confocal laser scanning microscopy, fluorescence in situ hybridization, and scanning electron microscopy. The study found that lipid emulsions could promote the formation of E. coli–C. albicans mixed-species BFs, especially with 10% lipid emulsions. The mechanism by which lipid emulsions promote mixed-species BF formation may involve significant upregulation of the expression of the flhDC, iha, HTA1, and HWP1 genes, which are associated with bacterial motility, adhesion, and BF formation. The results derived from this study necessitate strict aseptic precautions when handling lipid emulsions and avoiding the use of high concentrations of lipid emulsions for as long as possible.

Effect of environmental and nutritional conditions on the formation of single and mixed-species biofilms and their efficiency in cadmium removal

Remediation of contaminated water and wastewater using biosorption methods has attracted significant attention in recent decades due to its efficiency, convenience and minimised environmental effects. Bacterial biosorbents are normally deployed as a non-living powder or suspension. Little is known about the mechanisms or rates of bacterial attachment to surfaces and effect of various conditions on the biofilm development, as well as efficiency of living biofilms in the removal of heavy metals. In the present study, the effect of environmental and nutritional conditions such as pH, temperature, concentrations of phosphate, glucose, amino acid, nitrate, calcium and magnesium, on planktonic and biofilm growth of single and mixed bacterial cultures, were measured. Actinomyces meyeri, Bacillus cereus, Escherichia coli, Pseudomonas fluorescens strains were evaluated to determine the optimum biofilm growth conditions. The Cd(II) biosorption efficiencies of the mixed-species biofilm developed in the optimum growth condition, were investigated and modelled using Langmuir, Freundlich and Dubnin Radushkevich models. The biofilm quantification techniques revealed that the optimum concentration of phosphate, glucose, amino acid, nitrate, calcium and magnesium for the biofilm development were 25, 10, 1, 1.5, 5 and 0.5 g L−1, respectively. Further increases in the nutrient concentrations resulted in less biofilm growth. The optimum pH for the biofilm growth was 7 and alkaline or acidic conditions caused significant negative effects on the bacterial attachment and development. The optimum temperatures for the bacterial attachment to the surface were between 25 and 35 °C. The maximum Cd(II) biosorption efficiency (99%) and capacity (18.19 mg g−1) of the mixed-species biofilm, occurred on day 35 (Ci = 0.1 mg L−1) and 1 (Ci = 20 mg L−1) of biofilm growth, respectively. Modelling of the biosorption data revealed that Cd(II) removal by the living biofilm was a physical process by a monolayer of biofilm. The results of present study suggested that environmental and nutritional conditions had a significant effect on bacterial biofilm formation and its efficiency in Cd(II) removal.

Modeling of Interspecific Interaction of Yersinia pseudotuberculosis and Listeria monocytogenes in a Mixed-Species Biofilms with Microorganisms Isolated from Coastal Waters of the Sea of Japan.

We studied the possibility of formation of mixed-species biofilms by Yersinia pseudotuberculosis and Listeria monocytogenes with marine saprotrophic bacteria Flavobacterium sp. and Micrococcus luteus isolated from the coastal waters of the Sea of Japan in summer. Model experiments showed that Flavobacterium sp. and Micrococcus luteus can form both single- and mixed-species biofilms with the specified pathogenic bacteria thus stimulating their growth. This can contribute to the preservation of the pathogens in the marine environment.

Influence of phosphate dosing on biofilms development on lead in chlorinated drinking water bioreactors

Phosphate dosing is used by water utilities to prevent plumbosolvency in water supply networks. However, there is a lack of knowledge regarding biofilm formation on lead and plastic materials when phosphate concentrations are modified in drinking water systems. In this study, biofilms were grown over lead coupons and PVC tubes in bioreactors supplied with local drinking water treated to provide different phosphate doses (below 1, 1 and 2 mg/L) over a period of 28 days. A range of commercial iron pellets (GEH104 and WARP) were tested aiming to maintain phosphate levels below the average 1 mg/L found in drinking water. Changes in biofilm community structure in response to three different phosphate treatments were characterised by Illumina sequencing of the 16S rRNA gene for bacteria and the ITS2 gene for fungi. Scanning electron microscopy was used to visualise physical differences in biofilm development in two types of materials, lead and PVC. The experimental results from the kinetics of phosphate absorption showed that the GEH104 pellets were the best option to, in the long term, reduce phosphate levels while preventing undesirable turbidity increases in drinking water. Phosphate-enrichment promoted a reduction of bacterial diversity but increased that of fungi in biofilms. Overall, higher phosphate levels selected for microorganisms with enhanced capabilities related to phosphorus metabolism and heavy metal resistance. This research brings new insights regarding the influence of different phosphate concentrations on mixed-species biofilms formation and drinking water quality, which are relevant to inform best management practices in drinking water treatment.

Inhibitory effects of xylitol and sorbitol on Streptococcus mutans and Candida albicans biofilms are repressed by the presence of sucrose

ObjectiveAmong the preventive and therapeutic options available for dental caries, sugar alcohols (xylitol and sorbitol) have been widely promoted as oral healthcare products due to its perceived anticariogenic effect. However, the therapeutic efficacy of these sugar alcohols against Streptococcus mutans and Candida albicans in a sucrose supplemented environment, as found in disease-prone conditions in the oral cavity, has not been adequately investigated. MethodsSingle and mixed-species biofilm formation was evaluated in medium with different concentrations of xylitol, sorbitol with or without sucrose supplementation. Biofilm quantification methods such as crystal violet assay, XTT assay, CFU counting complemented with confocal and electron microscopic techniques were used. ResultsUnder sucrose-free conditions, xylitol and sorbitol demonstrated a significant dose-dependent inhibitory effect on S. mutans biofilms, whereas inhibitory effect on C. albicans biofilm was weak. The presence of 1 % sucrose in the environment diminished the inhibitory effect of both xylitol and sorbitol on S. mutans and C. albicans mono-species biofilms. Sucrose supplementation on pre-formed S. mutans biofilms also reduced the inhibitory effect of xylitol. Xylitol and sorbitol presence reduced mixed-species biofilm formation and altered the biofilm architecture and glucan production. However, sucrose supplementation reduced the inhibitory effect of sugar alcohols and enhanced the mixed-species biofilm formation. ConclusionsXylitol and sorbitol exerts an inhibitory effect on S. mutans and C. albicans biofilm formation and this inhibitory effect is repressed by the presence of sucrose.

Lactobacillus Plantarum 108 Inhibits Streptococcus mutans and Candida albicans Mixed-Species Biofilm Formation.

Streptococcus mutans is the principal biofilm forming oral pathogen associated with dental caries. Studies have shown that Candida albicans, a commensal oral fungus is capable of forming pathogenic mixed-species biofilms with S. mutans. The treatment of bacterial and fungal infections using conventional antimicrobial agents has become challenging due to the antimicrobial resistance of the biofilm mode of growth. The present study aimed to evaluate the efficacy of secretory components of Lactobacillus plantarum 108, a potentially promising probiotic strain, against S. mutans and C. albicans single and mixed-species biofilms. L. plantarum 108 supernatant inhibited S. mutans and C. albicans single-species biofilms as shown by XTT reduction assay, crystal violet assay, and colony forming units counting. The probiotic supernatant significantly inhibited the S. mutans and C. albicans mixed-species biofilm formation. The pre-formed mixed-species biofilms were also successfully reduced. Confocal microscopy showed poorly developed biofilm architecture in the probiotic supernatant treated biofilms. Moreover, the expression of S. mutans genes associated with glucosyltransferase activity and C. albicans hyphal specific genes (HWP1, ALS1 and ALS3) were down-regulated in the presence of the probiotic supernatant. Altogether, the data demonstrated the capacity of L. plantarum 108 supernatant to inhibit the S. mutans and C. albicans mixed-species biofilms. Herein, we provide a new insight on the potential of probiotic-based strategies to prevent bacterial-fungal mixed-species biofilms associated with dental caries.

Fighting Mixed-Species Microbial Biofilms With Cold Atmospheric Plasma.

Most biofilms in nature are formed by multiple microbial species, and such mixed-species biofilms represent the actual lifestyles of microbes, including bacteria, fungi, viruses (phages), and/or protozoa. Microorganisms cooperate and compete in mixed-species biofilms. Mixed-species biofilm formation and environmental resistance are major threats to water supply, food industry, and human health. The methods commonly used for microbial eradication, such as antibiotic or disinfectant treatments, are often ineffective for mixed-species biofilm consortia due to their physical matrix barrier and physiological interactions. For the last decade, an increasing number of investigations have been devoted to the usage of cold atmospheric plasma (CAP), which is produced by dielectric barrier discharges or plasma jets to prevent or eliminate microbial biofilms. Here, we summarized the production of CAP, the inactivation of microorganisms upon CAP treatment, and the microbial factors affecting the efficacy of CAP procedure. The applications of CAP as antibiotic alternative strategies for fighting mixed-species biofilms were also addressed.

Biofilm and Planktonic Bacteria Differentially Express a Small Molecule (<3kD), Heat‐Stable, Protease‐Resistant Factor that Affects Bovine Neutrophil Function in a NF‐Kappa β‐Independent Mechanism

IntroductionChronic bacterial infections and the associated inflammation have a significant impact in both veterinary and human medicine. Bacterial biofilms are a major cause of the persistent inflammation observed in chronic infections and nearly 80% of all infections involve biofilms. Biofilms are typically polymicrobial in composition and many of these mixed‐infections involve anaerobic bacteria. Recent evidence demonstrates that immune cells often disregard the signals released from biofilm bacteria, and ineffectual clearance of biofilms may explain the chronicity of this sort of infection. A mixed‐species model of anaerobic biofilm growth was developed to evaluate medically relevant biofilms and their interactions with bovine neutrophils.AimsTo generate mixed‐species bacterial biofilms composed of the two opportunistic pathogens Fusobacterium necrophorum and Porphyromonas levii, and to employ the in vitro system to investigate factors associated with biofilms that may limit neutrophil functional ability.MethodsMixed‐species biofilm formation was verified with SEM, CLSM and viable cell counts. Neutrophil oxidative burst was assessed with a fluorescent microplate assay for hydrogen peroxide. Chemotaxis was measured with a Transwell transmigration assay. Extracellular factors released from planktonic cultures and biofilms were assessed by size‐exclusion filtration, lipopolysaccharide (LPS) removal, protease and heat treatment. Mechanistic experiments to study neutrophil responses to these supernatants were conducted with specific inhibitors of function, such as NF‐κβ signaling (BAY‐11) and inflammasome activation (glyburide).ResultsNeutrophils exposed to planktonic bacterial supernatants showed significantly elevated oxidative (4‐fold increase in fluorescence) and chemotactic responses compared to neutrophils exposed to biofilm products from identical bacteria. LPS plays a significant role in the stimulation of neutrophils; in this experiment biofilms produced substantially more LPS than planktonic bacteria despite inducing less neutrophil response. Removal of LPS resulted in similar neutrophil responses to planktonic and biofilm supernatants.Size‐exclusion filtration of bacterial supernatants and subsequent exposure to neutrophils revealed that the active stimulatory molecule is below 3 kD and is released strictly from planktonic cultures. Intensive heat and protease treatment of the <3kD fractions did not alter neutrophil functional responses. Treatment with a NF‐κβ inhibitor demonstrated that neutrophils respond specifically to planktonic supernatants independently of NF‐κβ signaling.ConclusionsWe describe a heat‐stable, protease‐resistant, non‐LPS, small (<3kD) factor produced by in vitro planktonic cultures of F. necrophorum and P. levii that activates bovine neutrophils. Neutrophils did not exhibit the same oxidative and chemotactic responses upon exposure to in vitro generated biofilm supernatants and these differential responses are the result of a NF‐κβ independent mechanism, perhaps through an inflammasome‐mediated process.Support or Funding InformationFunding provided by the University of Calgary Grant 10002461 to DW Morck.

Antimicrobial activity of nitrochalcone and pentyl caffeate against hospital pathogens results in decreased microbial adhesion and biofilm formation

The present study investigated the antimicrobial, anti-adhesion and anti-biofilm activity of the modified synthetic molecules nitrochalcone (NC-E05) and pentyl caffeate (C5) against microorganisms which have a high incidence in hospital-acquired infections. The compounds were further tested for their preliminary systemic toxicity in vivo. NC-E05 and C5 showed antimicrobial activity, with minimum inhibitory concentrations (MICs) ranging between 15.62 and 31.25 μg ml−1. Treatment with NC-E05 and C5 at 1 × MIC and/or 10 × MIC significantly reduced mono or mixed-species biofilm formation and viability. At MIC/2, the compounds decreased microbial adhesion to HaCaT keratinocytes from 1 to 3 h (p < 0.0001). In addition, NC-E05 and C5 demonstrated low toxicity in vivo in the Galleria mellonella model at anti-biofilm concentrations. Thus, the chemical modification of these molecules proved to be effective in the proposed anti-biofilm activity, opening opportunities for the development of new antimicrobials.

The Significance of Lipids to Biofilm Formation in Candida albicans: An Emerging Perspective.

Candida albicans, the dimorphic opportunistic human fungal pathogen, is capable of forming highly drug-resistant biofilms in the human host. Formation of biofilm is a multistep and multiregulatory process involving various adaptive mechanisms. The ability of cells in a biofilm to alter membrane lipid composition is one such adaptation crucial for biofilm development in C. albicans. Lipids modulate mixed species biofilm formation in vivo and inherent antifungal resistance associated with these organized communities. Cells in C. albicans biofilms display phase-dependent changes in phospholipid classes and in levels of lipid raft formation. Systematic studies with genetically modified strains in which the membrane phospholipid composition can be manipulated are limited in C. albicans. In this review, we summarize the knowledge accumulated on the impact that alterations in phospholipids may have on the biofilm forming ability of C. albicans in the human host. This review may provide the requisite impetus to analyze lipids from a therapeutic standpoint in managing C. albicans biofilms.

Antibiofilm efficacy of curcumin in combination with 2-aminobenzimidazole against single- and mixed-species biofilms of Candida albicans and Staphylococcus aureus.

Mixed fungal and bacterial biofilm associated infections of implants have been a huge challenge in health care because of the increased resistance to antimicrobials and the critical biological differences between fungi and bacteria. In this study, we evaluated the 2-aminobenzimidazole (2ABI) and curcumin (CUR) alone to inhibit planktonic cell growth, adhesion as well as single and mixed species biofilms of Candida albicans and Staphylococcus aureus on silicone. The combined effects between 2ABI and CUR on mixed species biofilm formation and pre-formed biofilm were assessed. Our work showed that 2ABI or CUR alone was effective as a sole agent, inhibiting planktonic growth, adhesion and the biofilm formation of bacteria and fungi on the silicone surface. The combination of 2ABI and CUR exhibited the enhanced effect on mixed biofilm compared to mono-drug therapy. The biofilm architecture was investigated by scanning electron microscopy (SEM) and the distinction of living/dead organisms within biofilm was examined by confocal laser scanning microscopy (CLSM). The combination activity was most potent on mixed biofilm. These results suggest the potential applicability of 2ABI and CUR to treatment of biofilm related device infections.

Structure of a 1.5 MDa bacterial adhesin reveals its role in the mixed-species biofilm formation with diatoms on ice

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6 μm-long adhesin using a ‘dissect and build’ structural biology approach and have established complementary roles for its five distinct Regions. Domains in Region I (RI) tether the adhesin to the Type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical Ig-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligandbinding domains that join diatoms and bacteria together in a mixed species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain; and the terminal RV domain contains several ‘Repeats-in-Toxin’ motifs and a non-cleavable signal sequence that target proteins for export via the Type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity

Occurrence of Aerobic and Anaerobic Bacteria in the Consumer End of a Water Distribution System.

In this study, mixed species biofilm formation including sulphate-reducing bacteria (SRB) on polypropylene surface and bacteriology of network water were investigated in a model water distribution system during a nine-month period. Water and biofilm samples were analyzed for the enumeration of aerobic heterotrophic bacteria (AHB), anaerobic heterotrophic bacteria (ANHB) and SRB. The number of live/dead bacteria was also analyzed by epifluorescence microscopy. In addition, extracellular polysaccharide substances (EPS) extraction, carbohydrate analysis and scanning electron microscope observation were performed. A biofilm with heterogeneous structure formed on the polypropylene surface of the model water distribution system. Live/dead staining data indicated that biofilm matured in the first month. It was observed that especially AHB entered into a viable but not culturable state because of the temperature decrease. It was also noted that temperature is an important environmental factor especially for planktonic SRB. The quantity of carbohydrate significantly decreased according to the temperature.

Inhibition of multispecies biofilms by a fluoride-releasing dental prosthesis copolymer

ObjectivesThis study aimed to develop a new mixed-species acidogenic biofilm model and use it to assess the antimicrobial properties of a novel fluoride-releasing copolymer. MethodsStubs composed of a copolymer of methyl methacrylate (MMA) and 2-hydroxyethyl methacrylate (HEMA) with polymethyl methacrylate (PMMA) were produced by chemically-activated free radical polymerization. A fluoride-releasing copolymer was developed by incorporating sodium fluoride in place of a portion of the PMMA. Samples were mounted in polysulfone Modified Robbins Devices (MRDs) and were optimized for single- and mixed-species biofilm formation by Candida albicans, Lactobacillus casei and Streptococcus mutans. ResultsFluoride release was sustained for at least 48h in flowing conditions. Fluoride did not affect the colonization and biofilm growth of any of the microorganisms in monocultures. However, in mixed-species biofilms, cell densities of all three species were reduced approximately ten-fold (p<0.05) on the fluoridated material compared with the non-fluoridated copolymer. ConclusionsThese data demonstrate that intermicrobial interactions in mixed-species acidogenic biofilms are sensitive to fluoride, and that the inclusion of fluoride in a denture lining copolymer reduces the formation of polymicrobial biofilms. Clinical significanceThe growth of acidogenic microorganisms on denture materials is associated with denture stomatitis and dental caries on surrounding teeth. A fluoride-releasing copolymer that inhibits acidogenic mixed-species biofilms, such as the material described in this study, has the potential to control these diseases by limiting biofilm growth.

Évaluation du potentiel de formation de biofilms mixtes entre Candida albicans et quelques espèces bactériennes isolées de cathéters vasculaires périphériques au CHU de Tlemcen. Première étude en Algérie

Mixed-species biofilms constitute a reservoir of infection for a group of bacteria and yeasts that coexist on the same support. Peripheral venous catheters make up a good surface for the attachment of microorganisms that promote biofilm formation and this requires complex strategies for antimicrobial treatments. No such studies on formation mixed biofilms have ever been conducted in Algeria. Therefore, we evaluated the potential for the formation of mixed-species biofilms by Candida albicans and some bacterial species isolated from peripheral vascular catheters at the University Hospital of Tlemcen, in Algeria. The results obtained showed that C.albicans have the potential to form mixed biofilms with three bacteria (Enterobacter cloacae, Bordetella spp. and Serratia liquefaciens) isolated from the same catheter as the yeasts. The amount of biofilms produced varies depending on the species and the composition of the growth medium. Observations by scanning electron microscopy showed that the structure of the mixed biofilm depends on the surface support the biofilm was formed on, and varies with the species. A competition was noted between bacteria and yeasts; it depends on the composition of the medium and its pH, which both play an important role in promoting the dominance of one over the other.

Single- and mixed-species biofilm formation by Escherichia coli O157:H7 and Salmonella, and their sensitivity to levulinic acid plus sodium dodecyl sulfate

The development of single- and mixed-species biofilms formed by Escherichia coli O157:H7 and Salmonella was observed, and the antimicrobial effectiveness of levulinic acid (LVA) plus sodium dodecyl sulfate (SDS) on the cells in single- and dual-species biofilms was determined. Biofilm-forming ability of single- and mixed-species cultures was observed by crystal violet staining and their resistance to levulinic acid plus SDS was determined by enumeration. Fluorescent protein-labeled E. coli O157:H7 and Salmonella were constructed and the bacterial composition of the biofilms after treatment with levulinic acid plus SDS was visualized by confocal laser scanning microscopy (CLSM). E. coli O157:H7 and Salmonella were antagonistic to each other, being more sensitive to levulinic acid plus SDS in mixed-species biofilms. Images captured by CLSM revealed that E. coli O157:H7 and Salmonella were distributed evenly in the single- and dual-species biofilms, and confirmed that the combination of levulinic acid and SDS was effective in inactivating bacterial cells in biofilms. Results revealed that levulinic acid with SDS may be used as a potential biofilm control intervention.