Stages Of Biofilm Development Research Articles

Monitoring bacterial biofilms with a microfluidic flow chip designed for imaging with white-light interferometry.

There is a need for imaging and sensing instrumentation that can monitor transitions in a biofilm structure in order to better understand biofilm development and emergent properties such as anti-microbial resistance. Herein, we describe the design, manufacture, and use of a microfluidic flow cell to visualize the surface structure of bacterial biofilms with white-light interferometry (WLI). The novel imaging chip enabled the use of this non-disruptive imaging method for the capture of high resolution three-dimensional profile images of biofilm growth over time. The fine axial resolution (3 nm) and the wide field of view (>1 mm by 1 mm) enabled the detection of biofilm formation as early as 3 h after inoculation of the flow cell with a live bacterial culture (Pseudomonas fluorescens). WLI imaging facilitated the monitoring of the early stages of biofilm development and subtle variations in the structure of mature biofilms. Minimally-invasive imaging enabled the monitoring of biofilm structure with surface metrology metrics (e.g., surface roughness). The system was used to observe a transition in the biofilm structure that occurred in response to exposure to a common antiseptic. In the future, WLI and the biofilm imaging cell described herein may be used to test the effectiveness of biofilm-specific therapies to combat common diseases associated with biofilm formation such as cystic fibrosis and periodontitis.

Effects of meat juice on biofilm formation of Campylobacter and Salmonella

Campylobacter and Salmonella are leading causes of foodborne illnesses worldwide, vastly harboured by raw meat as their common food reservoir. Both microbes are prevalent in meat processing environments in the form of biofilms that contribute to cross-contamination and foodborne infection. This study applied raw meat juice (chicken juice and pork juice) as a minimally processed food model to study its effects on bacterial biofilm formation. Meat juice was collected during the freeze-thaw process of raw meat and sterilized by filtration. In 96-well polystyrene plates and glass chambers, supplementation of over 25% meat juice (v/v) in laboratory media led to an increase in biofilm formation of Campylobacter and Salmonella. During the initial attachment stage of biofilm development, more bacterial cells were present on surfaces treated with meat juice residues compared to control surfaces. Meat juice particulates on abiotic surfaces facilitated biofilm formation of Campylobacter and Salmonella under both static and flow conditions, with the latter being assessed using a microfluidic platform. Further, the deficiency in biofilm formation of selected Campylobacter and Salmonella mutant strains was restored in the presence of meat juice particulates. These results suggested that meat juice residues on the abiotic surfaces might act as a surface conditioner to support initial attachment and biofilm formation of Campylobacter and Salmonella. This study sheds light on a possible survival mechanism of Campylobacter and Salmonella in meat processing environments, and indicates that thorough cleaning of meat residues during meat production and handling is critical to reduce the bacterial load of Campylobacter and Salmonella.

Effectiveness of Devices to Monitor Biofouling and Metals Deposition on Plumbing Materials Exposed to a Full-Scale Drinking Water Distribution System.

A Modified Robbins Device (MRD) was installed in a full-scale water distribution system to investigate biofouling and metal depositions on concrete, high-density polyethylene (HDPE) and stainless steel surfaces. Bulk water monitoring and a KIWA monitor (with glass media) were used to offline monitor biofilm development on pipe wall surfaces. Results indicated that adenosine triphosphate (ATP) and metal concentrations on coupons increased with time. However, bacterial diversities decreased. There was a positive correlation between increase of ATP and metal deposition on pipe surfaces of stainless steel and HDPE and no correlation was observed on concrete and glass surfaces. The shared bacterial diversity between bulk water and MRD was less than 20% and the diversity shared between the MRD and KIWA monitor was only 10%. The bacterial diversity on biofilm of plumbing material of MRD however, did not show a significant difference suggesting a lack of influence from plumbing material during early stage of biofilm development.

Quantifying Biofilm Formation of <i>Sinorhizobium meliloti</i> Bacterial Strains in Microfluidic Platforms by Measuring the Diffusion Coefficient of Polystyrene Beads

Though the majority of bacteria can form structured communities known as biofilms, mutations can cause bacterial strains to vary in their ability to form a biofilm. In this study, the apparent diffusion coefficient of polystyrene microspheres 0.29 μm in diameter, which were executing Brownian motion inside bacterial colonies, was used as a quantitative parameter of the ability of a strain to form a biofilm and of the biofilm development. The study was performed using five Sinorhizobium meliloti strains, the biofilm-forming strains Rm8530 expR+, Rm8530 exoY, and Rm9034 expG, and the non-biofilm forming strains Rm1021 and Rm9030-2 expA1. The green fluorescent beads were placed with each strain in a separate channel of a microfluidic device. Thus, as the bacterial colonies grew under identical conditions over a 4-day period, the motion of the fluorescent microspheres was recorded and the diffusion coefficients were measured every 24 hours via particle tracking algorithms. It was found that each strain displayed a unique pattern of change in diffusion coefficient over time. Also, for a given biofilm-forming strain, there was a clear correlation between the value of the diffusion coefficient and the appearance and motility of the bacterial community. Thus, the diffusion coefficient can be used to identify different S. meliloti strains, and for the biofilm-forming strains, it is also a quantitative indicator of the stage of biofilm development.

Presence of Extracellular DNA during Biofilm Formation by Xanthomonas citri subsp. citri Strains with Different Host Range.

Xanthomonas citri subsp. citri (Xcc) A strain causes citrus bacterial canker, a serious leaf, fruit and stem spotting disease of several Citrus species. X. alfalfae subsp. citrumelonis (Xac) is the cause of citrus bacterial spot, a minor disease of citrus nursery plants and X. campestris pv. campestris (Xc) is a systemic pathogen that causes black rot of cabbage. Xanthomonas spp. form biofilms in planta that facilitate the host infection process. Herein, the role of extracellular DNA (eDNA) was evaluated in the formation and stabilization of the biofilm matrix at different stages of biofilm development. Fluorescence and light microscopy, as well as DNAse treatments, were used to determine the presence of eDNA in biofilms and bacterial cultures. DNAse treatments of Xcc strains and Xac reduced biofilm formation at the initial stage of development, as well as disrupted preformed biofilm. By comparison, no significant effect of the DNAse was detected for biofilm formation by Xc. DNAse effects on biofilm formation or disruption varied among Xcc strains and Xanthomonas species which suggest different roles for eDNA. Variation in the structure of fibers containing eDNA in biofilms, bacterial cultures, and in twitching motility was also visualized by microscopy. The proposed roles for eDNA are as an adhesin in the early stages of biofilm formation, as an structural component of mature bacterial aggregates, and twitching motility structures.

남극 해양에서 생물막 생성 초기 단계의 세균 군집 구조 변화

Compared to planktonic bacterial populations, biofilms have distinct bacterial community structures and play important ecological roles in various aquatic environments. Despite their ecological importance in nature, bacterial community structure and its succession during biofilm development in the Antarctic marine environment have not been elucidated. In this study, the succession of bacterial community, particularly during the early stage of biofilm development, in the Antarctic marine environment was investigated by pyrosequencing of the 16S rRNA gene. Overall bacterial distribution in biofilms differed considerably from surrounding seawater. Relative abundance of Gammaproteobacteria and Bacteroidetes which accounted for 78.9–88.3% of bacterial community changed drastically during biofilm succession. Gammaproteobacteria became more abundant with proceeding succession (75.7% on day 4) and decreased to 46.1% on day 7. The relative abundance of Bacteroidetes showed opposite trend to Gammaproteobacteria, decreasing from the early days to the intermediate days and becoming more abundant in the later days. There were striking differences in the composition of major OTUs (≥ 1%) among samples during the early stages of biofilm formation. Gammaproteobacterial species increased until day 4, while members of Bacteroidetes, the most dominant group on day 1, decreased until day 4 and then increased again. Interestingly, Pseudoalteromonas prydzensis was predominant, accounting for up to 67.4% of the biofilm bacterial community and indicating its important roles in the biofilm development.

Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity.

When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell-cell communication, microorganisms secrete molecules called quorum-sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast-to-hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

Salivary pellicles equalise surfaces’ charges and modulate the virulence of Candida albicans biofilm

IntroductionNumerous environmental factors influence the pathogenesis of Candida biofilms and an understanding of these is necessary for appropriate clinical management. AimsTo investigate the role of material type, pellicle and stage of biofilm development on the viability, bioactivity, virulence and structure of C. albicans biofilms. MethodsThe surface roughness (SR) and surface free energy (SFE) of acrylic and titanium discs was measured. Pellicles of saliva, or saliva supplemented with plasma, were formed on acrylic and titanium discs. Candida albicans biofilms were then generated for 1.5h, 24h, 48h and 72h. The cell viability in biofilms was analysed by culture, whilst DNA concentration and the expression of Candida virulence genes (ALS1, ALS3 and HWP1) were evaluated using qPCR. Biofilm metabolic activity was determined using XTT reduction assay, and biofilm structure analysed by Scanning Electron Microscopy (SEM). ResultsWhilst the SR of acrylic and titanium did not significantly differ, the saliva with plasma pellicle increased significantly the total SFE of both surface. The number of viable microorganisms and DNA concentration increased with biofilm development, not differing within materials and pellicles. Biofilms developed on saliva with plasma pellicle surfaces had significantly higher activity after 24h and this was accompanied with higher expression of virulence genes at all periods. ConclusionInduction of C. albicans virulence occurs with the presence of plasma proteins in pellicles, throughout biofilm growth. To mitigate such effects, reduction of increased plasmatic exudate, related to chronic inflammatory response, could aid the management of candidal biofilm-related infections.

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua.

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism.

Interkingdom cooperation between Candida albicans, Streptococcus oralis and Actinomyces oris modulates early biofilm development on denture material.

Candida-associated stomatitis affects up to 60% of denture wearers, and Candida albicans remains the most commonly isolated fungal species. The oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque. The aims of this study were to determine the effects of S. oralis and A. oris on the development of C. albicans biofilms on denture material. Resin discs were coated with saliva and at early (1.5 h) or later (24 h) stages of biofilm development, cell numbers of each species were determined. Spatial distribution of microorganisms was visualized by confocal scanning laser microscopy of biofilms labelled by differential fluorescence or by fluorescence in situ hybridization. Interkingdom interactions underpinning biofilm development were also evaluated planktonically utilizing fluorescence microscopy. Synergistic interactions between all three species occurred within biofilms and planktonically. Bacterial cells coaggregated with each other and adhered singly or in coaggregates to C. albicans hyphal filaments. Streptococcus oralis appeared to enhance hyphal filament production and C. albicans biovolume was increased 2-fold. Concomitantly, cell numbers of S. oralis and A. oris were enhanced by C. albicans. Thus, cooperative physical and metabolic processes occurring between these three microbial species intensify pathogenic plaque communities on denture surfaces.

Antimicrobial efficacy of different concentration of sodium hypochlorite on the biofilm of Enterococcus faecalis at different stages of development.

BackgroundPersistent infection of the root canal due to the presence of resistance bacterial species, such as Enterococcus faecalis, has always been one of the most important reasons for endodontic treatment failure. This study investigated the antimicrobial efficacy of 1%, 2.5 % and 5% sodium hypochlorite in eliminating E. faecalis biofilms at different stages of development.Material and MethodsIn this study 4-, 6- and 10-week-old E. faecalis biofilms were subjected to one of the following approaches: phosphate-buffered saline solution (PBS) or 1%, 2.5% and 5% NaOCl. Dentin chip suspensions were used for colony forming unit (CFU) counting to estimate remaining E. faecalis counts. Statistical comparison of the means was carried out with Kruskal-Wallis test, and pair-wise comparisons were made by Mann-Whitney U test, at a significance level of P<0.05.ResultsThe results showed that 2.5% and 5% NaOCl completely eliminated E. faecalis biofilms in three stages of biofilm development, whereas 1% NaOCl resulted in 85.73%, 81.88% and 78.62% decreases in bacterial counts in 4-, 6- and 10-week-old biofilms, respectively, which was significantly more than those with PBS (p<0.05).Conclusions The bacteria in mature and old biofilms were more resistant to 1% NaOCl than were the bacteria in young biofilms. Overall survival rate and residual bacteria increased with biofilm aging. Key words:Antibacterial, biofilm, E. faecalis, sodium hypochlorite.

Tracheal tube biofilm removal through a novel closed-suctioning system: an experimental study

Tracheal tube biofilm develops during mechanical ventilation. We compared a novel closed-suctioning system vs standard closed-suctioning system in the prevention of tracheal tube biofilm. Eighteen pigs, on mechanical ventilation for 76 h, with P. aeruginosa pneumonia were randomized to be tracheally suctioned via the KIMVENT* closed-suctioning system (control group) or a novel closed-suctioning system (treatment group), designed to remove tracheal tube biofilm through saline jets and an inflatable balloon. Upon autopsy, two tracheal tube hemi-sections were dissected for confocal and scanning electron microscopy. Biofilm area, maximal and minimal thickness were computed. Biofilm stage was assessed. Sixteen animals were included in the final analysis. In the treatment and control group, the mean (sd) pulmonary burden was 3.34 (1.28) and 4.17 (1.09) log cfu gr(-1), respectively (P=0.18). Tracheal tube P. aeruginosa colonization was 5.6 (4.9-6.3) and 6.2 (5.6-6.9) cfu ml(-1) (median and interquartile range) in the treatment and control group, respectively (P=0.23). In the treatment group, median biofilm area was 3.65 (3.22-4.21) log10 μm2 compared with 4.49 (4.27-4.52) log10 μm2 in the control group (P=0.031). In the treatment and control groups, the maximal biofilm thickness was 48.3 (26.7-71.2) µm (median and interquartile range) and 88.8 (43.8-125.7) µm, respectively. The minimal thickness in the treatment and control group was 0.6 (0-4.0) µm and 23.7 (5.3-27.8) µm (P=0.040) (P=0.017). Earlier stages of biofilm development were found in the treatment group (P<0.001). The novel CSS reduces biofilm accumulation within the tracheal tube. A clinical trial is required to confirm these findings and the impact on major outcomes.

Novel Insights into the Proteus mirabilis Crystalline Biofilm Using Real-Time Imaging.

The long-term use of indwelling catheters results in a high risk from urinary tract infections (UTI) and blockage. Blockages often occur from crystalline deposits, formed as the pH rises due to the action of urease-producing bacteria; the most commonly found species being Proteus mirabilis. These crystalline biofilms have been found to develop on all catheter materials with P. mirabilis attaching to all surfaces and forming encrustations. Previous studies have mainly relied on electron microscopy to describe this process but there remains a lack of understanding into the stages of biofilm formation. Using an advanced light microscopy technique, episcopic differential interference contrast (EDIC) microscopy combined with epifluorescence (EF), we describe a non-destructive, non-contact, real-time imaging method used to track all stages of biofilm development from initial single cell attachment to complex crystalline biofilm formation. Using a simple six-well plate system, attachment of P. mirabilis (in artificial urine) to sections of silicone and hydrogel latex catheters was tracked over time (up to 24 days). Using EDIC and EF we show how initial attachment occurred in less than 1 h following exposure to P. mirabilis. This was rapidly followed by an accumulation of an additional material (indicated to be carbohydrate based using lectin staining) and the presence of highly elongated, motile cells. After 24 h exposure, a layer developed above this conditioning film and within 4 days the entire surface (of both catheter materials) was covered with diffuse crystalline deposits with defined crystals embedded. Using three-dimensional image reconstruction software, cells of P. mirabilis were seen covering the crystal surfaces. EDIC microscopy could resolve these four components of the complex crystalline biofilm and the close relationship between P. mirabilis and the crystals. This real-time imaging technique permits study of this complex biofilm development with no risk of artefacts due to sample manipulation. A full understanding of the stages and components involved in crystalline encrustation formation will aid in the development of new protocols to manage and ultimately prevent catheter blockage.

Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms.

Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties but that also facilitate surface motility and influence biofilm formation. Detailed studies addressing the significance of lipopeptides for biofilm formation and architecture are rare. Hence, the present study sets out to determine the specific role of the lipopeptide viscosin in Pseudomonas fluorescens SBW25 biofilm formation, architecture and dispersal, and to relate viscA gene expression to viscosin production and effect. Initially, we compared biofilm formation of SBW25 and the viscosin-deficient mutant strain SBW25ΔviscA in static microtitre assays. These experiments demonstrated that viscosin had little influence on the amount of biofilm formed by SBW25 during the early stages of biofilm development. Later, however, SBW25 formed significantly less biofilm than SBW25ΔviscA. The indication that viscosin is involved in biofilm dispersal was confirmed by chemical complementation of the mutant biofilm. Furthermore, a fluorescent bioreporter showed that viscA expression was induced in biofilms 4 h prior to dispersal. Subsequent detailed studies of biofilms formed in flow cells for up to 5 days revealed that SBW25 and SBW25ΔviscA developed comparable biofilms dominated by well-defined, mushroom-shaped structures. Carbon starvation was required to obtain biofilm dispersal in this system. Dispersal of SBW25 biofilms was significantly greater than of SBW25ΔviscA biofilms after 3 h and, importantly, carbon starvation strongly induced viscA expression, in particular for cells that were apparently leaving the biofilm. Thus, the present study points to a role for viscosin-facilitated motility in dispersal of SBW25 biofilms.

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H2S) and methane (CH4) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H2S and CH4 production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H2S emission was notably high (290.7±72.3 mg S-H2S liter(-1) day(-1)), whereas emissions of CH4 remained low (17.9±15.9 mg COD-CH4 liter(-1) day(-1)). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH4 emissions increased after 1 year of biofilm growth (327.6±16.6 mg COD-CH4 liter(-1) day(-1)), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.

Sodium houttuyfonate inhibits biofilm formation and alginate biosynthesis-associated gene expression in a clinical strain of Pseudomonas aeruginosa in vitro.

The increasing multidrug resistance of Pseudomonas aeruginosa has become a serious public-health problem. In the present study, the inhibitory activities of sodium houttuyfonate (SH) against biofilm formation and alginate production in a clinical strain of P.aeruginosa (AH16) were investigated in vitro using crystal violet dying and standard curve methods, respectively. The cellular morphology of P. aeruginosa treated with SH was observed using a scanning electron microscope. Furthermore, reverse transcription-quantitative polymerase chain reaction was used to identify differences in the expression levels of genes associated with alginate biosynthesis as a result of the SH treatment. The results indicated that SH significantly inhibited biofilm formation, and decreased the levels of the primary biofilm constituent, alginate, in P. aeruginosa AH16 at various stages of biofilm development. In addition, scanning electron microscopy observations demonstrated that SH markedly altered the cellular morphology and biofilm structure of P. aeruginosa. Furthermore, the results from the reverse transcription-quantitative polymerase chain reaction analysis indicated that SH inhibited biofilm formation by mitigating the expression of the algD and algR genes, which are associated with alginate biosynthesis. Therefore, the present study has provided novel insights into the potent effects and underlying mechanisms of SH-induced inhibition of biofilm formation in a clinical strain of P. aeruginosa.

Nanofiltration and reverse osmosis surface topographical heterogeneities: Do they matter for initial bacterial adhesion?

The role of the physicochemical and surface properties of NF/RO membranes influencing bacterial adhesion has been widely studied. However, there exists a poor understanding of the potential role membrane topographical heterogeneities can have on bacterial adhesion. Heterogeneities on material surfaces have been shown to influence bacterial adhesion and biofilm development. The purpose of this study was therefore to investigate whether the presence of membrane topographical heterogeneities had a significant role during bacterial adhesion as this could significantly impact on how biofouling develops on membranes during NF/RO operation. An extensive study was devised in which surface topographical heterogeneities from two commercial membranes, NF270 and BW30, were assessed for their role in the adhesion of two model organisms of different geometrical shapes, Pseudomonas fluorescens and Staphylococcus epidermidis. The influence of cross-flow velocity and permeate flux was also tested, as well as the angle to which bacteria adhered was compared to the flow direction. Bacterial adhesion onto the membranes and in their surface topographical heterogeneities was assessed using Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), fluorescence microscopy and image analysis. Results showed that up to 30% of total adhered cells were found in membrane defect areas when defect areas only covered up to 13% of the membrane surface area. This suggests that topographical heterogeneities may play a significant role in establishing environmental niches during the early stages of biofilm development. Furthermore, no noticeable difference between the angle of cell attachment in defect areas compared to the rest of the membrane surface was found.

In vitro evaluation of the antibiofilm properties of chlorhexidine and delmopinol on dental implant surfaces

Biofilm development on the internal surfaces of dental implants and dental implant components may be associated with peri-implant hard and soft tissue abnormalities. Currently, no protocols have been established for effective disinfection of removable implant components. The purpose of this study was to evaluate the antibiofilm properties of commonly used dental antimicrobial agents (chlorhexidine and delmopinol) on multispecies biofilms. Biofilms of 48h and 168h maturity were exposed to 0.2% chlorhexidine, 2% chlorhexidine and a product containing 0.2% delmopinol for 5, 10 and 20min. 2% chlorhexidine was the most effective agent, achieving a total viable biofilm reduction ranging from 96.2% to >99.99% depending on the time of exposure and the stage of biofilm development.

Anthranilate deteriorates the structure of Pseudomonas aeruginosa biofilms and antagonizes the biofilm-enhancing indole effect.

Anthranilate and indole are alternative degradation products of tryptophan, depending on the bacterial species. While indole enhances the biofilm formation of Pseudomonas aeruginosa, we found that anthranilate, the tryptophan degradation product of P. aeruginosa, had an opposite effect on P. aeruginosa biofilm formation, in which anthranilate deteriorated the mushroom structure of biofilm. The anthranilate effect on biofilm formation was differentially exerted depending on the developmental stage and the presence of shear force. Anthranilate slightly accelerated the initial attachment of P. aeruginosa at the early stage of biofilm development and appeared to build more biofilm without shear force. But anthranilate weakened the biofilm structure in the late stage, deteriorating the mushroom structure of biofilms with shear force to make a flat biofilm. To investigate the interplay of anthranilate with indole in biofilm formation, biofilms were cotreated with anthranilate and indole, and the results showed that anthranilate antagonized the biofilm-enhancing effect of indole. Anthranilate was able to deteriorate the preformed biofilm. The effect of anthranilate and indole on biofilm formation was quorum sensing independent. AntR, a regulator of anthranilate-degrading metabolism was synergistically activated by cotreatment with anthranilate and indole, suggesting that indole might enhance biofilm formation by facilitating the degradation of anthranilate. Anthranilate slightly but significantly affected the cyclic diguaniylate (c-di-GMP) level and transcription of major extracellular polysaccharide (Psl, Pel, and alginate) operons. These results suggest that anthranilate may be a promising antibiofilm agent and antagonize the effect of indole on P. aeruginosa biofilm formation.

Quorum sensing triggers the stochastic escape of individual cells from Pseudomonas putida biofilms

The term ‘quorum sensing’ (QS) is generally used to describe the phenomenon that bacteria release and perceive signal molecules to coordinate cooperative behaviour in response to their population size. QS-based communication has therefore been considered a social trait. Here we show that QS signals (N-acyl-homoserine lactones, AHLs) are stochastically produced in young biofilms of Pseudomonas putida and act mainly as self-regulatory signals rather than inducing neighbouring cells. We demonstrate that QS induces the expression of putisolvin biosurfactants that are not public goods, thereby triggering asocial motility of induced cells out of microcolonies. Phenotypic heterogeneity is most prominent in the early stages of biofilm development, whereas at later stages behaviour patterns across cells become more synchronized. Our findings broaden our perspective on QS by showing that AHLs can control the expression of asocial (self-directed) traits, and that heterogeneity in QS can serve as a mechanism to drive phenotypic heterogeneity in self-directed behaviour.