Early Stages Of Biofilm Formation Research Articles

Inhibitory Effects and Mechanism of Action of Elsinochrome A on Candida albicans and Its Biofilm.

Biofilm-associated Candida albicans infections, the leading cause of invasive candidiasis, can cause high mortality rates in immunocompromised patients. Photodynamic antimicrobial chemotherapy (PACT) is a promising approach for controlling infections caused by biofilm-associated C. albicans. This study shows the effect of Elsinochrome A (EA) against different stages of C. albicans biofilms in vitro by XTT reduction assay and crystal violet staining. The mechanism of action of EA on C. albicans biofilm was analyzed with flow cytometry, confocal laser microscopy, and the Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). EA-mediated PACT significantly reduced the viability of C. albicans, with an inhibition rate on biofilm of 89.38% under a concentration of 32 μg/mL EA. We found that EA could not only inhibit the adhesion of C. albicans in the early stage of biofilm formation, but that it also had good effects on pre-formed mature biofilms with a clearance rate of 35.16%. It was observed that EA-mediated PACT promotes the production of a large amount of reactive oxygen species (ROS) in C. albicans and down-regulates the intracellular expression of oxidative-stress-related genes, which further disrupted the permeability of cell membranes, leading to mitochondrial and nuclear damage. These results indicate that EA has good photodynamic antagonizing activity against the C. albicans biofilm, and potential clinical value.

D-alanylation of lipoteichoic acid contributes to biofilm formation and acidogenesis capacity of Streptococcusmutans

BackgroundD-alanylation of Lipoteichoic acid (LTA) is considered to be essential for virulence factors expression in Gram-positive microorganism. The effects of the D-alanylation of LTA on biofilm formation and acidogenesis of Streptococcus mutans (S. mutans) are still not clearly understood. AimThis study was designed to investigate the impact of D-alanylation of LTA on biofilm formation and acidogenesis of S. mutans and explore the related mechanisms. Methods and materialWe compared the biofilm formation process by fluorescence microscope observation of LTA D-alanylation blocking strain with that of the wildtype strain. Auto-aggregation, cell surface charge, and polysaccharide production assays were performed to investigate the related mechanisms. pH drop assay and glycolysis pH drop-down analysis were carried out to evaluate the acidogenesis capacity of S. mutans after LTA D-alanylation blocking. To identify the biofilm formation and adhesive-related genes expressions of S. mutans mutant, qRT-PCR was performed. ResultsAfter blocking off the D-alanylation of LTA, S. mutans could not form the three-dimensional structural biofilm, in which cells were scattered on the substratum as small clusters. The auto-aggregation was prompted due to the mutant strain cell morphology change (*p < 0.05). Furthermore, more negative charges were found on the mutant strain cells surfaces and fewer water-insoluble glucans were produced in mutant biofilm (*p < 0.05). The adhesion capacity of the S. mutans biofilm was impaired after LTA D-alanylation blocking (*p < 0.05). Biofilm formation and adhesive-related genes expressions decreased (*p < 0.05), especially at the early stages of biofilm formation. S. mutans mutant strains exhibited suppressed acidogenesis because its glycolytic activity was impaired. ConclusionThe results of this study suggest that blocking of LTA D-alanylation disrupts normal biofilm formation in S. mutans predominantly if not entirely by altering intercellular auto-aggregation, cell adhesion, and extracellular matrix formation. Moreover, our study results suggest that the LTA D-alanylation plays an important role in S. mutans acidogenesis by altering glycolytic activity. These findings add to the knowledge about mechanisms underlying biofilm formation and acid tolerance in S. mutans.

Toward Real-Time Determination of Yield Coefficients of Early-Stage Electroactive Biofilms by Optical Microscopy

Physiological cellular parameters, such as latency times (lagt), cell production rates (CPR), doubling times (td), relative growth rates (RGR), and yield coefficients (YNe), are only known as endpoint measurements for electroactive microorganisms (EAM). Here we show that these can be gained non-invasively and in real-time for early-stage biofilm formation at electrodes using a microfluidic electrochemical flow-cell (EFC) allowing in vivo optical microscopy. Parameters obtained for early-stage mixed culture biofilm anodes formed at +150 mV vs. Ag/AgCl sat. KCl have lagt of 2.31–4.58 days, CPR of 0.72–1.20 × 105 cells h−1, td of 176.54–1838.65 min, RGR of 0.02–0.27 h−1, and YNe of 5.99–7.94 × 1012 cells mole-−1. However, oxygen permeation into the EFC was the main problem that remained unsolved during the study that provides interesting lessons for future improvements.

Colistin conditioning surfaces combined with antimicrobial treatment to prevent ventilator-associated infections

Biofilm formation on endotracheal tubes (ETT) is an important factor in the development of ventilator-associated pneumonia (VAP). This work aimed to investigate the effectiveness of colistin (COL) against the early stages of biofilm formation by Pseudomonas aeruginosa. Two strategies were used: pre-conditioning the adhesion surfaces with COL before biofilm formation and growing biofilms in its presence. The combined effect of treating P. aeruginosa 24-hours old biofilms with Ciprofloxacin (CIP) or colistin (COL) on clean and COL-conditioned surfaces was also assessed. Random deposition of COL residues altered the physico-chemical properties of the adhesion surfaces and impaired biofilm formation. Moreover, as a consequence of the reduced amount of biofilms attached to COL conditioned surfaces, adhered cells became more exposed to the subsequent action of CIP or COL, suggesting a combined outcome of prophylactic and therapeutic COL-based strategies. Results highlighted the promising use of COL to prevent the establishment of biofilms on ETT.

Anti-biofilm activities of coumarin as quorum sensing inhibitor for Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is a keystone pathogen in periodontitis, a biofilm-mediated infection disease. This research aimed to investigate the effect of coumarin on P. gingivalis biofilm formation. We detected the antimicrobial effect on P. gingivalis planktonic growth, observed membrane structure and morphological change by TEM, and quantified membrane permeability by calcein-AM staining. The cell surface hydrophobicity, aggregation, and attachment were assessed. We also investigated different sub-MIC concentrations of coumarin on biofilm formation, and observed biofilm structureby confocal laser scanning microscopy. The biofilm-related gene expression was evaluated using real-time PCR. The results showed that coumarin inhibited P. gingivalis growth and damaged the cell morphology above 400 μM concentration. Coumarin did not affect cell surface hydrophobicity, aggregation, attachment, and the early stage of biofilm formation at sub-MIC concentrations. Still, it exhibited anti-biofilm effects for the late-stage and pre-formed biofilms dispersion. The biofilms after coumarin treatment became interspersed, and biofilm-related gene expression was downregulated. Coumarin also inhibited AI-2 activity and interacted with the HmuY protein by molecular docking analysis. Our research demonstrated that coumarin inhibited P. gingivalis biofilm formation through a quorum sensing system.

Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim.

Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation. This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.

Collagen Peptide in a Combinatorial Treatment with Lactobacillus rhamnosus Inhibits the Cariogenic Properties of Streptococcus mutans: An In Vitro Study.

Dental caries is caused by the formation of cariogenic biofilm, leading to localized areas of enamel demineralization. Streptococcus mutans, a cariogenic pathogen, has long been considered as a microbial etiology of dental caries. We hypothesized that an antagonistic approach using a prebiotic collagen peptide in combination with probiotic Lactobacillus rhamnosus would modulate the virulence of this cariogenic biofilm. In vitro S. mutans biofilms were formed on saliva-coated hydroxyapatite discs, and the inhibitory effect of a combination of L. rhamnosus and collagen peptide on S. mutans biofilms were evaluated using microbiological, biochemical, confocal imaging, and transcriptomic analyses. The combination of L. rhamnosus with collagen peptide altered acid production by S. mutans, significantly increasing culture pH at an early stage of biofilm formation. Moreover, the 3D architecture of the S. mutans biofilm was greatly compromised when it was in the presence of L. rhamnosus with collagen peptide, resulting in a significant reduction in exopolysaccharide with unstructured and mixed bacterial organization. The presence of L. rhamnosus with collagen peptide modulated the virulence potential of S. mutans via down-regulation of eno, ldh, and atpD corresponding to acid production and proton transportation, whereas aguD associated with alkali production was up-regulated. Gly-Pro-Hyp, a common tripeptide unit of collagen, consistently modulated the cariogenic potential of S. mutans by inhibiting acid production, similar to the bioactivity of a collagen peptide. It also enhanced the relative abundance of commensal streptococci (S. oralis) in a mixed-species biofilm by inhibiting S. mutans colonization and dome-like microcolony formation. This work demonstrates that food-derived synbiotics may offer a useful means of disrupting cariogenic communities and maintaining microbial homeostasis.

Efficiency of a Novel Multifunctional Corrosion Inhibitor Against Biofilms Developed on Carbon Steel.

In natural environments, populations of microorganisms rapidly colonise surfaces forming biofilms. These sessile communities comprise a variety of species which contribute to biofouling and microbiologically influenced corrosion (MIC), especially on metals. Species heterogeneity in natural systems confers higher tolerance to adverse conditions such as biocide treatment compared with single species laboratory simulations. Effective chemical treatments to combat recalcitrant biofilms are often dangerous to apply; both to operators and the environment, and face international embargoes. Today, there is a drive to exchange current toxic and environmentally hazardous biocides with less harmful compounds. One effective method of achieving this goal is to generate multi-functional compounds capable of tackling corrosion and biofilm formation simultaneously, thus reducing the number of compounds in dosing procedures. In a previous study, a novel corrosion inhibitor demonstrated biocidal effects against three marine isolates during the early stages of biofilm formation. The compound; CTA-4OHcinn, holds great promise as a multi-functional inhibitor, however its effect on complex, multi-species biofilms remains unknown. Here we evaluate CTA-4OHcinn biocidal capacity against multi-species biofilms developed from oilfield samples. Mature biofilms were developed and treated with 10 mM CTA-4OHcinn for 4 h. The effects of the compound were assessed using mean probable number (MPN), adenosine triphosphate (ATP) quantification, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results demonstrate that CTA-4OHcinn significantly reduces the viability of mature biofilms, supporting previous demonstrations on the secondary function of CTA-4OHcinn as a biocide. CLSM results further indicate that CTA-4OHcinn targets the cell membrane resulting in lysis. This finding complements the established corrosion inhibition function of CTA-4OHcinn, indicating the compound is a true multi-functional organic corrosion inhibitor.

Early Stage Biofilm Formation on Bio-Based Microplastics in a Freshwater Reservoir

Early Stage Biofilm Formation on Bio-Based Microplastics in a Freshwater Reservoir

Thickness and roughness of transparent gold-palladium anodes have no impact on growth kinetics and yield coefficients of early-stage Geobacter sulfurreducens biofilms

Geobacter sulfurreducens is the model organism for electroactive microorganisms performing direct extracellular electron transfer and forming thick mature biofilm electrodes. Although numerous physiological properties of mature biofilm electrodes are deciphered, there is an extensive gap of knowledge on the early-stage biofilm formation. We have shown recently that transparent gold–palladium (AuPd) electrodes allow for analysis of early-stage biofilm formation using confocal laser scanning microscopy. Here we analysed the influence of thickness (ranging from 12.5 to 200 nm) and roughness of AuPd electrodes on physiological parameters of G. sulfurreducens early-stage biofilms. We show that when grown potentiostatically at −200 mV vs. Ag/ AgCl sat. KCl neither maximum current density (jmax of ∼ 80-150 µA cm−2) nor lag time (lag t of ∼ 0.2-0.4 days) or single cell yield coefficients (YNe of 1.43 × 1012 cells mole-−1) of the biofilms are influenced by the electrode preparation. This confirms the robustness of the experimental approach, which is an inevitable prerequisite for obtaining reliable results in follow-up experiments.

Effect of sublethal concentrations of bactericidal antibiotics on mutation frequency and stress response of Listeria monocytogenes

The purpose of this study was to investigate sublethal concentrations (SLC) of bactericidal antibiotics (ampicillin, gentamicin, kanamycin, and vancomycin) on the mutation frequency and stress response of antibiotic-induced-mutated (AIM) Listeria monocytogenes. Three L. monocytogenes strains (reference, clinical, and food isolate strains) were used in this study. SLC of bactericidal antibiotics significantly increased the mutation frequency in L. monocytogenes. It was found that AIM L. monocytogenes had a superior biofilm-forming ability than nontreated L. monocytogenes. This result correlated with the amounts of EPS produced (polysaccharide and protein) in the early stage of biofilm formation. AIM L. monocytogenes showed strong viability under food-associated stress (thermal, osmotic, and acidic) compared to nontreated L. monocytogenes. In addition, expression levels of motility (flaA) and virulence genes (hlyA, actA, and prfA) of AIM L. monocytogenes were significantly downregulated in the reference strain but significantly upregulated or similar to the expression levels in the clinical and food isolate strains compared to nontreated L. monocytogenes. Based on our results, SLC of bactericidal antibiotics increased the mutation frequency in L. monocytogenes, facilitated the adaptation of the bacterium to food-associated stress, and led to an increase in its pathogenicity.

Assessment of phage-mediated control of antibiotic-resistant Salmonella Typhimurium during the transition from planktonic to biofilm cells

This study was designed to evaluate the abilities of phage P22 to lyse, eradiate, and disperse the biofilm cells of Salmonella enterica serovar Typhimurium ATCC 19585 (STWT), ciprofloxacin-induced Typhimurium ATCC 19585 (STCIP), S. Typhimurium KCCM 40253 (STKCCM), and multidrug-resistant S. Typhimurium CCARM 8009 (STCCARM) in association with hydrophobicity, auto-aggregation, motility, protein content, extracellular DNA, and depolymerase activity. The affinity to hexadecane was significantly increased in STWT, STKCCM, and STCCARM cells after P22 infection. All strains tested showed relatively higher auto-aggregation abilities in the presence of P22 than the absence of P22. STKCCM showed the greatest auto-aggregative ability (23%) in the presence of P22, while STWT showed the least auto-aggregative ability (9%) in the absence of P22. The bacterial swimming motility affected the bacterial attachment at the early stage of biofilm formation. The red, dry and rough morphotype was observed for all strains tested. The numbers of STWT, STCIP, and STKCCM planktonic cells were considerably reduced by 7.2, 5.0, and 5.0 log CFU/ml, respectively, and STWT, STCIP, and STKCCM biofilm-forming cells were reduced by 5.8, 4.5, and 4.9 log, respectively, after 24 h of phage infection. The depolymerase produced by phages were confirmed by the presence of outer rim of plaques. Phages could be considered as promising alternatives for the control of biofilms due to their advantages including enzymatic degradation of extracellular biofilm matrix. The study would provide useful information for understanding the dynamic interactions between phages and biofilms and also designing the effective phage-based control system as an alterative strategy against biofilms.

Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni.

Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species, naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated ([Formula: see text]luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

Water absorption coefficient drives Nostoc sp. colonization on mineral substrates

Nostoc is a genus of filamentous Cyanobacteria common in both terrestrial and aquatic habitats. On land, Nostoc species can also form biofilms on stone monuments, due to their ability to remain desiccated for months or years, and fully recover metabolic activity within hours to days after rehydration with liquid water. We performed in vitro experiments inoculating a Nostoc sp. strain collected from the UNESCO site of archaeological park of Pompeii on five different stone materials, namely tuff, brick, limestone, porphyry, and glass paste to assess the role of water availability on early stage of biofilm formation on stones. The stone tiles, incubated in sealed glass chambers at four different relative humidity values (100%, 92%, 85%, 76%), were observed weekly in a 12-weeks interval to determine surface colonization and growth rate. Confocal laser scanning microscopy (CLSM) was then used to assess thickness, cellular volume and exopolysaccharides (EPS) production on each material. We found that in humidity driven in vitro colonization of Nostoc sp. water absorption coefficient (WAC) determines the propagation of the colonies; in particular, tuff and brick showed the best bioreceptivity, and a greater Nostoc sp. colonization rate, which was attributed to the higher values of WAC.

Mussel Adhesive Mimetic Silk Sericin Prepared by Enzymatic Oxidation for the Construction of Antibacterial Coatings.

With the rapid development and advancement in orthodontic and orthopedic technologies, the demand for biomedical-grade titanium (Ti) alloys is growing. The Ti-based implants are susceptible to bacterial infections, leading to poor healing and osteointegration, resulting in implant failure or repeated surgical intervention. Silk sericin (SS) is hydrophilic, biocompatible, and biodegradable and could induce a low immunological response in vivo. As a result, it would be intriguing to investigate the use of hydrophilic SS in surface modification. In this work, the tyrosine moiety in SS was oxidized by tyrosinase (or polyphenol oxidase) to the 3,4-dihydroxyphenylalanine (DOPA) form, generating the catechol moiety-containing SS (SSC). Inspired by the adhesion of mussel foot proteins, the SSC coatings could be directly deposited onto multiple surfaces in SS and tyrosinase mixed stock solutions to create active surfaces with catechol groups. Further, the SSC-coated Ti surfaces were hybridized with silver nanoparticles (Ag NPs) via in situ silver ion (Ag+) reduction. The antibacterial properties of the Ag NPs/SS-coated Ti surfaces are demonstrated, and they can prevent bacterial cell adhesion as well as early-stage biofilm formation. In addition, the developed Ag NPs/SSC-coated Ti surfaces exhibited a negligible level of cytotoxicity in L929 mouse fibroblast cells.

Transcriptome Analysis Reveals the Genes Involved in Bifidobacterium Longum FGSZY16M3 Biofilm Formation.

Biofilm formation has evolved as an adaptive strategy for bacteria to cope with harsh environmental conditions. Currently, little is known about the molecular mechanisms of biofilm formation in bifidobacteria. A time series transcriptome sequencing analysis of both biofilm and planktonic cells of Bifidobacterium longum FGSZY16M3 was performed to identify candidate genes involved in biofilm formation. Protein–protein interaction network analysis of 1296 differentially expressed genes during biofilm formation yielded 15 clusters of highly interconnected nodes, indicating that genes related to the SOS response (dnaK, groS, guaB, ruvA, recA, radA, recN, recF, pstA, and sufD) associated with the early stage of biofilm formation. Genes involved in extracellular polymeric substances were upregulated (epsH, epsK, efp, frr, pheT, rfbA, rfbJ, rfbP, rpmF, secY and yidC) in the stage of biofilm maturation. To further investigate the genes related to biofilm formation, weighted gene co-expression network analysis (WGCNA) was performed with 2032 transcript genes, leading to the identification of nine WGCNA modules and 133 genes associated with response to stress, regulation of gene expression, quorum sensing, and two-component system. These results indicate that biofilm formation in B. longum is a multifactorial process, involving stress response, structural development, and regulatory processes.

Atomic Layer Deposition of ZrO2 on Titanium Inhibits Bacterial Adhesion and Enhances Osteoblast Viability.

PurposeThe study was intended to create a uniform zirconia layer even on the surface of complex structures via atomic layer deposition (ALD). The impact of crystalline zirconia deposited by ALD on bacterial adhesion and osteoblast viability was assessed via surface treatment of dental implants.MethodsAmorphous zirconia was deposited using an atomic layer deposition reactor (Atomic Classic, CN1, Hwaseong, Korea) on titanium discs. Heating the samples at 400°C resulted in crystallization. Samples were divided into three groups: the control group, the group carrying amorphous ALD-zirconia (Z group), and the heat-treated group following zirconia ALD deposition (ZH group).The surface of each sample was analyzed, followed by the assessment of adhesion of Streptococcus mutans and Porphyromonas gingivalis, and viability and differentiation of MC3T3-E1 cells.ResultsThe adhesion of S. mutans and P. gingivalis was significantly reduced in the Z and ZH groups compared with the control group (P < 0.05). The viability of MC3T3-E1 cells was significantly increased in the ZH group compared with the control group (P < 0.001), while no significant differences were observed in the Z group (P > 0.05). Differentiation of MC3T3-E1 cells showed a marginally significant increase in the ZH group compared with the control group (P < 0.1), while no significant differences were found in the Z group (P > 0.1).ConclusionCompared with the pure titanium group, the groups that were coated with zirconia via ALD showed a decreased adhesion of S. mutans during the early stages of biofilm formation and P. gingivalis adhesion inducing peri-implantitis, and an increase in MC3T3-E1 cell viability and differentiation. The findings indicate the possibility of treating the implant surface to reduce peri-implantitis and improve osseointegration.

Early and differential bacterial colonization on microplastics deployed into the effluents of wastewater treatment plants

Microbial colonization of microplastics (MPs) in aquatic ecosystems is a well-known phenomenon; however, there is insufficient knowledge of the early colonization phase. Wastewater treatment plant (WWTP) effluents have been proposed as important pathways for MPs entry and transport in aquatic environments and are hotspots of bacterial pathogens and antibiotic resistance genes (ARGs). This study aimed at characterizing bacterial communities in the early stage of biofilm formation on seven different types of MPs deployed in two different WWTPs effluents as well as measuring the relative abundance of two ARGs (sulI and tetM) on the tested MPs. Illumina Miseq sequencing of the 16S rRNA showed significant higher diversity of bacteria on MPs in comparison with free-living bacteria in the WWTP effluents. β-diversity analysis showed that the in situ environment (sampling site) and hydrophobicity, to a lesser extent, had a role in the early bacterial colonization phase. An early colonization phase MPs-core microbiome could be identified. Furthermore, specific core microbiomes for each type of polymer suggested that each type might select early attachment of bacteria. Although the tested WWTP effluent waters contained antibiotic resistant bacteria (ARBs) harboring the sulI and tetM ARGs, MPs concentrated ARBs harboring the sulI gene but not tetM. These results highlight the relevance of the early attachment phase in the development of bacterial biofilms on different types of MP polymers and the role that different types of polymers might have facilitating the attachment of specific bacteria, some of which might carry ARGs.

Structural Diversity in Early-Stage Biofilm Formation on Microplastics Depends on Environmental Medium and Polymer Properties

Plastics entering the environment can not only undergo physical degradation and fragmentation processes, but they also tend to be colonized by microorganisms. Microbial colonization and the subsequent biofilm formation on plastics can alter their palatability to organisms and result in a higher ingestion as compared to pristine plastics. To date, the early stage of biofilm formation on plastic materials has not been investigated in context of the environmental medium and polymer properties. We explored the early-stage biofilm formation on polyamide (PA), polyethylene terephthalate (PET), and polyvinyl chloride (PVC) after incubation in freshwater and artificial seawater and categorized the structural diversity on images obtained via scanning electron microscopy. Furthermore, by the measurement of the initial ζ-potential of the plastic materials, we found that PA with the highest negative ζ-potential tended to have the highest structural diversity, followed by PET and PVC after incubation in freshwater. However, PVC with the lowest negative ζ-potential showed the highest structural diversity after incubation in seawater, indicating that the structural diversity is additionally dependent on the incubation medium. Our results give insights into how the incubation medium and polymer properties can influence the early-stage biofilm formation of just recently environmentally exposed microplastics. These differences are responsible for whether organisms may ingest microplastic particles with their food or not.

Cross-Kingdom Cell-to-Cell Interactions in Cariogenic Biofilm Initiation

Candida albicans is known to form polymicrobial biofilms with various Streptococcus spp., including mitis and mutans group streptococci. Streptococcus gordonii (mitis group) has been shown to bind avidly to C. albicans hyphae via direct cell-to-cell interaction, while the cariogenic pathogen Streptococcus mutans (mutans group) interacts with the fungal cells via extracellular glucans. However, the biophysical properties of these cross-kingdom interactions at the single-cell level during the early stage of biofilm formation remain understudied. Here, we examined the binding forces between S. mutans (or S. gordonii) and C. albicans in the presence and absence of in situ glucans on the fungal surface using single-cell atomic force microscopy and their influence on biofilm initiation and subsequent development under cariogenic conditions. The data show that S. gordonii binding force to the C. albicans surface is significantly higher than that ofS. mutans to the fungal surface (~2-fold). However, S. mutans binding forces are dramatically enhanced when the C. albicans cell surface is locally coated with extracellular glucans (~6-fold vs. uncoated C. albicans), which vastly exceeds the forces between S. gordonii andC. albicans. The enhanced binding affinity of S. mutans to glucan-coated C. albicans resulted in a larger structure during early biofilm initiation compared to S. gordonii–C. albicans biofilms. Ultimately, this resulted in S. mutans dominance composition in the 3-species biofilm model under cariogenic conditions. This study provides a novel biophysical aspect of Candida-streptococcal interaction whereby extracellular glucans may selectively favor S. mutans binding interactions with C. albicans during cariogenic biofilm development.