Phases Of Biofilm Development Research Articles

UV stabilizers can foster early development of biofilms on freshwater microplastics

Interactions between microbes and microplastics are important as of emerging plastic loads in the global environment. Although diverse plastic additives are used in large amounts, there are very few studies on a quantitative comparison of plastisphere on plastics with different plastic additives. We studied the effects of two widely used UV stabilizers (benzotriazole-type UV-327 and benzophenone-type UV-531 were selected based on their persistence and toxicity) in low-density polyethylene (LDPE) on freshwater microbes. This is the first study on the sole effects of UV stabilizers used as plastic additives on freshwater in situ plastisphere biofilm development. Confocal laser scanning microscopy, assisted with proper differentiating fluorochromes and threshold-based 3D segmentation of data, was used to visualize and quantify biofilm. On the first week of biofilm growth, there was very little biovolume and a negligible amount of phototrophs on pristine LDPE contrasting other substrates. Biovolumes were significantly higher on LDPE with UV stabilizers (up to 159% higher than pristine LDPE), although the biomass was mostly dead due to toxicity (>100% higher dead biovolume than live biovolume in LDPE with UV stabilizers). After the fourth week, marginally higher biovolumes along with a revival of the biomass on LDPE with UV stabilizers were observed. The ability to induce microorganismic intracellular reactive oxygen species by UV stabilizers was detected, which may stimulate biofilm growth during the primary phase of biofilm development. Atomic force microscopy analysis denoted that LDPE with UV stabilizers exhibit considerably stronger adhesion force than pristine LDPE. These observations suggest that UV stabilizers can foster the early attachment of microbes to microplastics while killing the surface contacting layer. An alive upper layer of microbes can get developed on the dead biofilm without much disruption due to the toxicity of UV stabilizers. This occurrence can eventually boost the early development of biofilms on plastics.

The role of immobilized quorum sensing strain in promoting biofilm formation of Moving Bed Biofilm Reactor during long-term stable operation

Quorum sensing (QS) signaling plays a significant role in the natural regulation of biofilm formation. Multiple species QS systems in wastewater treatment processes have received significant attention in recent years and this study presents a long-term analysis of QS signaling, bacterial structures and extracellular polymeric substance (EPS) during biofilm formation, detachment and reformation processes. Six types of Acyl homoserine lactones (AHLs) were found to be closely related to different phases of biofilm development, with both QS and quorum quenching (QQ) strains being identified as drivers of various biofilm phases and 10 strains presenting a close relationship with AHLs (p < 0.05). Meanwhile, QS strain Sphingomonas rubra was immobilized and added into reactor systems, resulting in significant increase in AHL content, EPS production, and adhesion strength of biofilm (p < 0.05), which might promote biofilm formation processes during long-term stable operation. This study provides a potentially simple and economical way to improve activity and stability of MBBR in complex wastewater systems.

Bacterial Abundance, Diversity and Activity During Long-Term Colonization of Non-biodegradable and Biodegradable Plastics in Seawater.

The microorganisms living on plastics called “plastisphere” have been classically described as very abundant, highly diverse, and very specific when compared to the surrounding environments, but their potential ability to biodegrade various plastic types in natural conditions have been poorly investigated. Here, we follow the successive phases of biofilm development and maturation after long-term immersion in seawater (7 months) on conventional [fossil-based polyethylene (PE) and polystyrene (PS)] and biodegradable plastics [biobased polylactic acid (PLA) and polyhydroxybutyrate-co-hydroxyvalerate (PHBV), or fossil-based polycaprolactone (PCL)], as well as on artificially aged or non-aged PE without or with prooxidant additives [oxobiodegradable (OXO)]. First, we confirmed that the classical primo-colonization and growth phases of the biofilms that occurred during the first 10 days of immersion in seawater were more or less independent of the plastic type. After only 1 month, we found congruent signs of biodegradation for some bio-based and also fossil-based materials. A continuous growth of the biofilm during the 7 months of observation (measured by epifluorescence microscopy and flow cytometry) was found on PHBV, PCL, and artificially aged OXO, together with a continuous increase in intracellular (3H-leucine incorporation) and extracellular activities (lipase, aminopeptidase, and β-glucosidase) as well as subsequent changes in biofilm diversity that became specific to each polymer type (16S rRNA metabarcoding). No sign of biodegradation was visible for PE, PS, and PLA under our experimental conditions. We also provide a list of operational taxonomic units (OTUs) potentially involved in the biodegradation of these polymers under natural seawater conditions, such as Pseudohongiella sp. and Marinobacter sp. on PCL, Marinicella litoralis and Celeribacter sp. on PHBV, or Myxococcales on artificially aged OXO. This study opens new routes for a deeper understanding of the polymers’ biodegradability in seawaters, especially when considering an alternative to conventional fossil-based plastics.

Ecological Linkages between a Biofilm Ecosystem and Reactor Performance: The Specificity of Biofilm Development Phases.

In biofilm-based engineered ecosystems, the reactor performance was closely linked to interspecies interactions within a biofilm ecosystem, whereas the ecological processes underpinning such linkage were still unenlightened. Herein, the principles of community succession and assembly were integrated to capture the ecological laws of biofilm development by molecular ecological networks and assembly model analysis based on the 16S rRNA sequencing analysis and metagenomics in a well-controlled moving bed biofilm reactor. At the initial colonization phase (days 0-2, driven by initial colonizers), interspecific cooperation (74.18%) facilitated initial biofilm formation, whereas some pioneers, and keystone species disappeared at later phases. At the accumulation phase (days 3-30, rapid biofilm development), interspecific cooperation (81.41 ± 5.07%) contributed to rapid biofilm development and keystone species were mainly involved in quorum sensing or positively correlated with extracellular polymeric substance production. At the maturation phase (days 31-106, a well-adapted quasi-equilibrium state), increased interspecific competition (32.74 ± 4.77%) and higher small-world property facilitated the rapid information transportation and pollutant treatment, and keystone species were positively correlated with the removal of COD and NH4+-N. Homogenizing dispersal diminished the contemporary community dissimilarities, while turnover but rather nestedness governed the temporal variations in the biofilm succession period. This study highlighted the specificity of ecological processes at distinct biofilm development phases, which would advance our understanding on the development-to-function linkages in biofilm-based treatment processes.

Influence of light and flow on taxon composition and photosynthesis of marine phototrophic biofilm in photobioreactors

In this study, marine biofilms were cultured in a flow-lane, semi-continuous photobioreactor at different irradiances and flows to evaluate their combined effect on biofilms' phototrophic composition and photosynthetic activity. Taxon richness, evaluated by different microscopy techniques, including transmission and scanning electron microscopy, resulted to be heavily reduced from source communities to mature cultures. The strongest decrease was observed for diatoms, which were overcome by cyanobacteria and green algae over time. Photosynthetic performance was investigated by pulse amplitude modulated fluorescence. Irradiance was the main driver of data distribution of the photosynthetic parameters rel.ETRmax and Ik, while flow rate affected α and ΔF/Fm’. The combination of irradiance and flow rate affected ΔF/Fm’ reflecting the photosynthetic performance of the most relatively abundant taxa. Higher ΔF/Fm’ was attained when cyanobacteria and green algae were dominating, whilst lower ΔF/Fm’ when diatoms occurred in the initial phase of biofilm development.

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint.

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization—12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.

The exopolysaccharide\u2013eDNA interaction modulates 3D architecture of Bacillus subtilis biofilm

BackgroundBacterial biofilms are surface-adherent microbial communities in which individual cells are surrounded by a self-produced extracellular matrix of polysaccharides, extracellular DNA (eDNA) and proteins. Interactions among matrix components within biofilms are responsible for creating an adaptable structure during biofilm development. However, it is unclear how the interactions among matrix components contribute to the construction of the three-dimensional (3D) biofilm architecture.ResultsDNase I treatment significantly inhibited Bacillus subtilis biofilm formation in the early phases of biofilm development. Confocal laser scanning microscopy (CLSM) and image analysis revealed that eDNA was cooperative with exopolysaccharide (EPS) in the early stages of B. subtilis biofilm development, while EPS played a major structural role in the later stages. In addition, deletion of the EPS production gene epsG in B. subtilis SBE1 resulted in loss of the interaction between EPS and eDNA and reduced the biofilm biomass in pellicles at the air-liquid interface. The physical interaction between these two essential biofilm matrix components was confirmed by isothermal titration calorimetry (ITC).ConclusionsBiofilm 3D structures become interconnected through surrounding eDNA and EPS. eDNA interacts with EPS in the early phases of biofilm development, while EPS mainly participates in the maturation of biofilms. The findings of this study provide a better understanding of the role of the interaction between eDNA and EPS in shaping the biofilm 3D matrix structure and biofilm formation.

The Transcription Factor Stp2 Is Important for Candida albicans Biofilm Establishment and Sustainability

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. The sensing and metabolism of amino acids play important roles during various phases of biofilm development – from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2Δ deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2Δ biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4, the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms.

Start-up of a nutrient removal system using Scenedesmus vacuolatus and Chlorella vulgaris biofilms

Microalgal biofilm-based technologies are of strong interest due to their high biomass concentrations and ability to remove nutrients from wastewater, utilize CO2 and produce secondary valuable products. This study investigated the biomass production and nutrient removal efficiency of the microalgae Scenedesmus vacuolatus ACUF_053 and Chlorella vulgaris ACUF_809 from a synthetic wastewater, describing a start-up process in a new biofilm photobioreactor (PBR) configuration. Two sets of experiments were performed. The first one compared the performance of a suspended and attached cell system under batch conditions. The second set of experiments was addressed under semi-batch conditions to study the microalgae biofilm development in the PBR. Five stages in the development of the biofilm were identified for S. vacuolatus: attachment, biofilm formation, maturation I, adaptation and maturation II. The biofilm development phases had a different nutrient removal efficiency. S. vacuolatus biofilm showed a higher phosphate demand during the first attachment and formation phases, while it had a higher nitrate demand during the subsequent phases. C. vulgaris biofilm formation was affected by the pH increase (up to 10.6). The biofilm PBR design using both S. vacuolatus and C. vulgaris showed potential for wastewater treatment due the higher nutrient removal rates.

A putative LysR-type transcriptional regulator inhibits biofilm synthesis in Pseudomonas aeruginosa

Biofilm formation is an important virulence factor which is controlled by complex regulatory circuits in Pseudomonas aeruginosa. In this work, a biofilm hyper-producing strain, P2-7, was selected from a collection of transposon insertion mutants in which the PA2121 gene was disrupted. PA2121 was predicted as a putative LysR-type regulator. Analyses showed that it was involved in early biofilm formation, mature biofilm development, and colony morphology. Quantitative measurements revealed that PA2121 repressed biosynthesis of extracellular polysaccharides (alginate, psl and pel). Furthermore, it was observed that PA2121 was self-regulated, highly expressed in the early phase of biofilm development, and subject to the negative regulation by a biofilm synthesis regulator SrpA that binds directly to the PA2121 gene promoter. Collectively, this study proposes that PA2121 is a novel biofilm synthesis repressor (BsrA) in P. aeruginosa.

Continuous shear stress alters metabolism, mass-transport, and growth in electroactive biofilms independent of surface substrate transport

Electroactive bacteria such as Geobacter sulfurreducens and Shewanella onedensis produce electrical current during their respiration; this has been exploited in bioelectrochemical systems. These bacteria form thicker biofilms and stay more active than soluble-respiring bacteria biofilms because their electron acceptor is always accessible. In bioelectrochemical systems such as microbial fuel cells, corrosion-resistant metals uptake current from the bacteria, producing power. While beneficial for engineering applications, collecting current using corrosion resistant metals induces pH stress in the biofilm, unlike the naturally occurring process where a reduced metal combines with protons released during respiration. To reduce pH stress, some bioelectrochemical systems use forced convection to enhance mass transport of both nutrients and byproducts; however, biofilms’ small pore size limits convective transport, thus, reducing pH stress in these systems remains a challenge. Understanding how convection is necessary but not sufficient for maintaining biofilm health requires decoupling mass transport from momentum transport (i.e. fluidic shear stress). In this study we use a rotating disc electrode to emulate a practical bioelectrochemical system, while decoupling mass transport from shear stress. This is the first study to isolate the metabolic and structural changes in electroactive biofilms due to shear stress. We find that increased shear stress reduces biofilm development time while increasing its metabolic rate. Furthermore, we find biofilm health is negatively affected by higher metabolic rates over long-term growth due to the biofilm’s memory of the fluid flow conditions during the initial biofilm development phases. These results not only provide guidelines for improving performance of bioelectrochemical systems, but also reveal features of biofilm behavior. Results of this study suggest that optimized reactors may initiate operation at high shear to decrease development time before decreasing shear for steady-state operation. Furthermore, this biofilm memory discovered will help explain the presence of channels within biofilms observed in other studies.

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

Streptococcus mutans-derived exopolysaccharides are virulence determinants in the matrix of biofilms that cause caries. Extracellular DNA (eDNA) and lipoteichoic acid (LTA) are found in cariogenic biofilms, but their functions are unclear. Therefore, strains of S. mutans carrying single deletions that would modulate matrix components were used: eDNA – ∆lytS and ∆lytT; LTA – ∆dltA and ∆dltD; and insoluble exopolysaccharide – ΔgtfB. Single-species (parental strain S. mutans UA159 or individual mutant strains) and mixed-species (UA159 or mutant strain, Actinomyces naeslundii and Streptococcus gordonii) biofilms were evaluated. Distinct amounts of matrix components were detected, depending on the inactivated gene. eDNA was found to be cooperative with exopolysaccharide in early phases, while LTA played a larger role in the later phases of biofilm development. The architecture of mutant strains biofilms was distinct (vs UA159), demonstrating that eDNA and LTA influence exopolysaccharide distribution and microcolony organization. Thus, eDNA and LTA may shape exopolysaccharide structure, affecting strategies for controlling pathogenic biofilms.

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development.

Two-component signaling is a specialized mechanism that bacteria use to respond to changes in their environment. Nonpathogenic strains of Escherichia coli K-12 harbor 30 histidine kinases and 32 response regulators, which form a network of regulation that integrates many other global regulators that do not follow the two-component signaling mechanism, as well as signals from central metabolism. The output of this network is a multitude of phenotypic changes in response to changes in the environment. Among these phenotypic changes, many two-component systems control motility and/or the formation of biofilm, sessile communities of bacteria that form on surfaces. Motility is the first reversible attachment phase of biofilm development, followed by a so-called swim or stick switch toward surface organelles that aid in the subsequent phases. In the mature biofilm, motility heterogeneity is generated by a combination of evolutionary and gene regulatory events.

Treatment of microbial biofilms in the post-antibiotic era: prophylactic and therapeutic use of antimicrobial peptides and their design by bioinformatics tools

The treatment for biofilm infections is particularly challenging because bacteria in these conditions become refractory to antibiotic drugs. The reduced effectiveness of current therapies spurs research for the identification of novel molecules endowed with antimicrobial activities and new mechanisms of antibiofilm action. Antimicrobial peptides (AMPs) have been receiving increasing attention as potential therapeutic agents, because they represent a novel class of antibiotics with a wide spectrum of activity and a low rate in inducing bacterial resistance. Over the past decades, a large number of naturally occurring AMPs have been identified or predicted from various organisms as effector molecules of the innate immune system playing a crucial role in the first line of defense. Recent studies have shown the ability of some AMPs to act against microbial biofilms, in particular during early phases of biofilm development. Here, we provide a review of the antimicrobial peptides tested on biofilms, highlighting their advantages and disadvantages for prophylactic and therapeutic applications. In addition, we describe the strategies and methods for de novo design of potentially active AMPs and discuss how informatics and computational tools may be exploited to improve antibiofilm effectiveness.

Optimal colonization and growth of marine benthic diatoms on artificial substrata: protocol for a routine use in bioindication

Benthic diatoms growing on hard substrata are used for their bioindication ability in freshwater quality monitoring. Artificial substrata are needed in cases where any natural substrate is present or to achieve similar sampling conditions between sites. Prior to use marine benthic diatoms for monitoring, a standardized protocol for sampling on artificial substrata must be set up. Two major types of information are required: (1) the time needed for a diatom community to be well developed and mature (climax stage); (2) the optimal growth conditions, given that the substrataum nature and texture are important parameters for the initial phase of biofilm development and can influence the future diatom assemblage. Three substrataum types were tested: frosted Plexiglass®, frosted glass, and rough enameled tiles. They were submerged for 8 weeks and sampled weekly. The experiment was conducted at five sites of distinct morphology and water chemistry, along the coastal area of Martinique Island, French West Indies. Development of diatom community was studied through biofilm dry weight, valve density, species richness, and species relative abundances. Globally, substratum type had no significant effect on any parameter. Frosted Plexiglass® was found to be the most interesting substratum because of higher valve densities and practical use. The asymptotic phase of biofilm development was encountered between 5 and 8 weeks depending on site and parameter. A compromise between community development and vandalism or loss through time was fixed to 5 weeks. This period is longer than for stream environments and is valid for tropical oligotrophic marine environments.

Novel Role for the Streptococcus pneumoniae Toxin Pneumolysin in the Assembly of Biofilms

ABSTRACTStreptococcus pneumoniae is an important commensal and pathogen responsible for almost a million deaths annually in children under five. The formation of biofilms by S. pneumoniae is important in nasopharyngeal colonization, pneumonia, and otitis media. Pneumolysin (Ply) is a toxin that contributes significantly to the virulence of S. pneumoniae and is an important candidate as a serotype-independent vaccine target. Having previously demonstrated that a luxS knockout mutant was unable to form early biofilms and expressed less ply mRNA than the wild type, we conducted a study to investigate the role of Ply in biofilm formation. We found that Ply was expressed in early phases of biofilm development and localized to cellular aggregates as early as 4 h postinoculation. S. pneumoniae ply knockout mutants in D39 and TIGR4 backgrounds produced significantly less biofilm biomass than wild-type strains at early time points, both on polystyrene and on human respiratory epithelial cells, cultured under static or continuous-flow conditions. Ply’s role in biofilm formation appears to be independent of its hemolytic activity, as S. pneumoniae serotype 1 strains, which produce a nonhemolytic variant of Ply, were still able to form biofilms. Transmission electron microscopy of biofilms grown on A549 lung cells using immunogold demonstrated that Ply was located both on the surfaces of pneumococcal cells and in the extracellular biofilm matrix. Altogether, our studies demonstrate a novel role for pneumolysin in the assembly of S. pneumoniae biofilms that is likely important during both carriage and disease and therefore significant for pneumolysin-targeting vaccines under development.

Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists

Protozoan grazing is a major mortality factor faced by bacteria in the environment. Vibrio cholerae, the causative agent of the disease cholera, is a natural inhabitant of aquatic ecosystems, and its survival depends on its ability to respond to stresses, such as predation by heterotrophic protists. Previous results show that grazing pressure induces biofilm formation and enhances a smooth to rugose morphotypic shift, due to increased expression of Vibrio polysaccharide (VPS). In addition to negatively controlling vps genes, the global quorum sensing (QS) regulator, HapR, plays a role in grazing resistance as the ΔhapR strain is efficiently consumed while the wild type (WT) is not. Here, the relative and combined contributions of VPS and QS to grazing resistance were investigated by exposing VPS and HapR mutants and double mutants in VPS and HapR encoding genes at different phases of biofilm development to amoeboid and flagellate grazers. Data show that the WT biofilms were grazing resistant, the VPS mutants were less resistant than the WT strain, but more resistant than the QS mutant strain, and that QS contributes to grazing resistance mainly in mature biofilms. In addition, grazing effects on biofilms of mixed WT and QS mutant strains were investigated. The competitive fitness of each strain in mixed biofilms was determined by CFU and microscopy. Data show that protozoa selectively grazed the QS mutant in mixed biofilms, resulting in changes in the composition of the mixed community. A small proportion of QS mutant cells which comprised 4% of the mixed biofilm biovolume were embedded in grazing resistant WT microcolonies and shielded from predation, indicating the existence of associational protection in mixed biofilms.

Inhibition of Staphylococcus epidermidis biofilm by trimethylsilane plasma coating.

Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections and promotes local progressive tissue destruction. Staphylococcus epidermidis infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which in combination with the increasing prevalence of antibiotic resistance among human pathogens further complicates treatment of biofilm-related device infections. We have developed a novel plasma coating technology. Trimethylsilane (TMS) was used as a monomer to coat the surfaces of 316L stainless steel and grade 5 titanium alloy, which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased S. epidermidis biofilm formation by inhibiting the attachment of bacterial cells to the TMS-coated surfaces during the early phase of biofilm development. We also discovered that bacterial cells on the TMS-coated surfaces were more susceptible to antibiotic treatment than their counterparts in biofilms on uncoated surfaces. These findings suggested that TMS coating could result in a surface that is resistant to biofilm development and also in a bacterial community that is more sensitive to antibiotic therapy than typical biofilms.

Effect of disinfectants on biofilm development by five species of Candida

Candida biofilms have become an increasingly significant clinical problem for hospital-acquired and device-associated infections due to their increased levels of resistance to the antifungals and disinfectants. This study examines the biofilm formation of fiveCandida isolates (Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei and Candida tropicalis), using the XTT (2, 3-Bis [2-methoxy- 4-nitro-5-(sulfenylamino) carbonyl-2H-tetrazolium-hydroxide]), and compares the susceptibility of biofilm cells to five disinfectants (glutaraldehyde, hydrogen peroxide, peracetic acid, ortho-phtalaldehyde and sodium hypochlorite) at the different times of biofilm formation (6th, 12th, 24th and 48th). Thus, the aim of our study was to investigate whether these disinfectants can interfere on Candida biofilm development phases. Our results indicated that the many variations in biofilm formation in clinical Candida species following disinfectant stress, however, none of the disinfectants completely removed the biofilm. Also, our transmission electron microscopic findings suggest that the disinfectants showed an important effect on the planktonic Candida cell. All these results also emphasize the importance of regular disinfection, before the starting of biofilm formation. On the other hand, XTT assay may not be the only adequate method to evaluate the efficacy of disinfectants. For these reasons, further studies are necessary to evaluate the disinfectant effectiveness on Candida biofilms. Key words: Biofilm, Candida, tetrazolium salt (XTT), disinfectant, , transmission electron microscope (TEM).

Antimicrobial Efficacy of Chlorhexidine against Bacteria in Biofilms at Different Stages of Development

Introduction Detailed knowledge on the nature of the physiological and metabolic phases of biofilm development is important in combating resistant, disease-associated biofilms. The aim of this study was to examine the susceptibility of multispecies biofilms at different phases of growth to root canal irrigants. Methods The multispecies biofilms were grown from plaque bacteria on collagen-coated hydroxyapatite discs in brain-heart infusion broth for time periods ranging from 2 days to several months. Fresh nutrients were added weekly for the first 3 weeks, followed by a nutrient deprivation phase, by adding fresh brain-heart infusion broth medium only once a month. Biofilms of different age were subjected to 1-, 3-, or 10-minute exposure to 2% chlorhexidine (CHX) or CHX-Plus. After treatment, the volume ratio of dead bacteria in biofilms was assessed by confocal laser scanning microscopy by using a LIVE/DEAD viability stain. Biofilm structure was visualized by scanning electron microscopy. Results The thickness of biofilms increased from 57 μm (2 days) to 155 μm (3 weeks) during biofilm development. It reached a steady state under nutrient-limiting conditions, with thickness of 190 μm (6 weeks) to 201 μm (12 weeks). The proportion of killed bacteria in mature biofilms (3 weeks) was lower than in young biofilms (2 days, 1 and 2 weeks) after treatment with both CHX products ( P < .01).The resistance of mature biofilms under the nutrient-limiting phase (6–12 weeks) to CHX remained stable and was similar to 3-week-old biofilm. However, treatment with CHX-Plus for 3 and 10 minutes did not lose effectiveness against biofilm bacteria in mature and nutrient-limited phases. CHX-Plus showed higher levels of bactericidal activity at all exposure times than 2% CHX ( P < .01). Conclusions Bacteria in mature biofilms and nutrient-limited biofilms are more resistant to CHX killing than in young biofilms. The results emphasize the importance of standardization of factors such as biofilm age when studying the comparative effectiveness of disinfecting agents against biofilm bacteria.