Young Biofilms Research Articles

Previously unknown regulatory role of extracellular RNA on bacterial directional migration.

Bacterial directional migration plays a significant role in bacterial adaptation. However, the regulation of this process, particularly in young biofilms, remains unclear. Here, we demonstrated the critical role of extracellular RNA as part of the Universal Receptive System in bacterial directional migration using a multidisciplinary approach, including bacterial culture, biochemistry, and genetics. We found that the destruction or inactivation of extracellular RNA with RNase or RNA-specific antibodies in the presence of the chemoattractant triggered the formation of bacterial "runner cells» in what we call a "panic state" capable of directional migration. These cells quickly migrated even on the surface of 1.5% agar and formed evolved colonies that were transcriptionally and biochemically different from the ancestral cells. We have also shown that cell-free DNA from blood plasma can act as a potent bacterial chemoattractant. Our data revealed a previously unknown role of bacterial extracellular RNA in the regulation of bacterial migration and have shown that its destruction or inhibition triggered the directional migration of developing and mature biofilms towards the chemoattractant.

Characterizing the development of biofilm in polyethylene pipes in the non-chlorinated Danish drinking-water distribution system

In newly commissioned drinking-water polyethylene (PE) pipes, biofilm develops on the inner pipe surface. The microbial community composition from colonization to the establishment of mature biofilms is less known, including the effect on the distributed water quality. Biofilm development was followed through 1.5 years in PE-pipe side streams at two locations of a full-scale, non-chlorinated drinking-water distribution system (leaving a waterworks versus 5–6 km from a waterworks) along with inlet and outlet water quality. Mature biofilms were established after ∼8–9 months, dominated by Proteobacteria, Actinobacteria and Saccharibacteria (61–93% relative abundance), with a higher diversity (OTUs/Shannon Index/16S rRNA gene amplicon sequencing) in pipes in the far end of the distribution system. Comamonadaceae, and specifically Aquabacterium (>30% of reads), dominated young (∼1.5-month-old) biofilms. Young biofilms were linked to increased microbiological counts in drinking water (HPC/ATP/qPCR), while the establishment of mature biofilms led to a drop in HPC and benefited the water quality, highlighting the importance of optimizing commissioning procedures for rapidly achieving mature and stable biofilms.

Increased susceptibility to azithromycin of Pseudomonas aeruginosa biofilms using RPMI 1640 testing media.

Azithromycin (AZM) is efficient for treatment of chronic Pseudomonas aeruginosa biofilm lung infections, despite of resistance in conventional susceptibility testing. It has been shown that planktonic P. aeruginosa are more susceptible to AZM when tested in RPMI 1640 medium. The aim of the study was to test the susceptibility to AZM of P. aeruginosa biofilms in LB vs RPMI 1640 media. We investigated the effect of AZM on planktonic and biofilms of (WT) P. aeruginosa (PAO1), the hypermutable (ΔmutS) and the antibiotic-resistant phenotype(ΔnfxB) mutants. The effect of AZM on young and mature biofilms was investigated in the modified Calgary Biofilm Device by estimation of the minimal biofilm inhibitory concentration (MBIC). The AZM MBIC90 in LB/RPMI1640 on young biofilms treated for 24 h was 16/4 μg/mL for PAO1, 32/8 μg/mL for ΔmutS, and 256/16 μg/mL for ΔnfxB, while in mature biofilms was 256/2 μg/mL for PAO1 and ΔmutS and 16/1 μg/mL for ΔnfxB. The effect of AZM was improved when the treatment was prolonged to 72 h, supporting the intracellular accumulation of AZM. An increased susceptibility of P. aeruginosa biofilms to AZM was observed in RPMI 1640 than in LB medium. Our results might improve susceptibility testing and dosing of AZM for treatment of biofilm infections.

Biofilm Disruption and Virulence Attenuation Effects of Essential Oil From Endemic Algerian Cistus munbyi (Cistaceae) Against Clinical Strains of Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an opportunistic, resistant and highly pathogenic bacteria, responsible for severe nosocomial infections in humans. There is need to search new antimicrobials to target P. aeruginosa strains. Cistus munbyi is used in traditional medicine to manage infections. Objectives: This work aims to investigate the antimicrobial, antibiofilm and other antivirulence effects of essential oil from C. munbyi against P. aeruginosa. Materials and methods: Essential oil (EO) was prepared by hydro-distillation from C. munbyi, characterized by GC–MS and evaluated for its antimicrobial, antibiofilm and anti-virulence effects against clinical isolates of P. aeruginosa. Results: 100% of compounds were identified with Terpinen-4-ol (33.20%), Sabinene (13.20%), α-Thujene (11.30%), and p-Cymene (9.94%) being the major constituents. The EO had good antimicrobial activity determined by diameters of inhibition zones and broth dilution. At minimal inhibitory concentration (MIC), the EO eliminated more that 70% of young biofilms in 19 strains out of 24 tested strains while at 2MIC, only one strain had biofilm inhibition below 70%. The EO eliminated more than 50% of mature biofilms in 13 strains out of 24 tested. Biofilm disruption was concentration-dependent at sub-MIC and indicated good potential of C. munbyi EO as P. aeruginosa antibiofilm agent. Scanning electron microscopy (SEM) images revealed that untreated colonies had a well-developed biofilm while there was significant reduction of biofilms with distorted architecture and cell shrinkage upon treatment with EO. The C. munbyi EO at MIC and sub-MIC was able to reduce quorum-sensing (QS) virulence factors such as pyocyanin production, exopolysaccharides matrix and elastase secretion. Significant QS revealed by reduction of violacein in C. violaceum (CV12472 and CV026) was observed. Conclusion: The overall results show good anti-pseudomonal effects of C. munbyi EO which makes it a potential agent to reduce the spread of infections and resistance to antibiotics from P. aeruginosa strains.

Effects of Sodium Hypochlorite Concentration and Application Time on Bacteria in an Ex Vivo Polymicrobial Biofilm Model

IntroductionIn endodontic treatment, it is important to remove or inactivate biofilms in the root canal system. We investigated the effects of different concentrations and application times of sodium hypochlorite (NaOCl) on the viability of bacteria in ex vivo polymicrobial biofilms of different maturation levels. MethodsPolymicrobial biofilms were prepared from dental plaque samples and grown for 1, 2, and 3 weeks under anaerobic conditions on collagen-coated hydroxyapatite discs as an ex vivo biofilm model. The biofilms were then exposed to NaOCl at concentrations ranging from 0.1% to 2% for 1 or 3 minutes. The control group was exposed to sterile distilled water. Viability staining was performed and examined by confocal laser scanning microscopy to determine the percentage of biofilm bacteria killed by NaOCl. Scanning electron microscopy was also performed to visually examine the biofilms. ResultsApplication of NaOCl at 0.5%–2% for both 1 and 3 min killed significantly more bacteria when compared to the controls (P < .05). Cell viability tended to be lower after the application of NaOCl for 3 minutes than that for 1 minute. ConclusionsOur experiments using an ex vivo model showed that within the range of 0.1%–2% of NaOCl, higher NaOCl concentrations and longer application times were more effective in killing biofilm bacteria, and that mature biofilms were more resistant to NaOCl than younger biofilms.

Mango peel as a potential enzyme inducer in Trichoderma harzianum

Water-insoluble exopolysaccharides (I-EPS) are a virulence factor for dental biofilms. It has already been demonstrated that mango pulp induces the secretion of glucan-hydrolytic enzymes in the fungus Trichoderma harzianum, and that they have an effect on I-EPS from young biofilms. Aim: Evaluate the effect of mango peel as an enzyme inducer in T. harzianum, and the effect of enzymes secreted on mature biofilms. Methods: Fractions of the peel (PL) and ethanol-precipitated pulp (PP) of Tommy Atkins mangoes were sterilized and added to a culture medium containing T. harzianum for induction of hydrolytic enzymes. After 192 h, the culture medium was centrifuged and the supernatant (enzyme extract) was used as treatment on S. mutans biofilms (n=9): a) NaCl 0.9 %; b) 0.12 % chlorhexidine digluconate; and c) extract of enzymes induced by PL or PP. Acidogenicity, bacterial viability, quantification of insoluble polysaccharides, and three-dimensional analysis of the biofilm by scanning electron microscopy (SEM) was performed. Data were analyzed by ANOVA followed by the Tukey test (α=5 %). Results: The hydrolytic enzymes did not alter the metabolism or bacterial viability of the biofilm (p&lt;0.05). Although the images obtained by SEM suggest some degree of matrix degradation, the quantification of I-EPS for the PL and PP groups did not differ from the control group (p&gt;0.05), suggesting a slight effect on the disorganization of the mature S. mutans biofilm. Conclusion: The results suggest that mango peel fraction can induce secretion of mutanase by T. harzianum, however in an insufficient amount to generate significant degradation on cariogenic biofilm.

Modelling drinking water biofilms: Bacterial adhesion and Legionella pneumophila necrotrophic growth

We propose a mathematical model to simulate the establishment and growth of a drinking water distribution system biofilm, focusing on the influence of ionic strength on bacterial adhesion and persistence of Legionella pneumophila. Specifically, we consider how ionic strength affects interaction energies during the initial phase of biofilm formation and we include the dependence of the attachment flux, modelled as a linear rate with respect to free floating cells, on it. The model also incorporates a novel necrotrophic kinetics to simulate the Legionella pneumophila metabolism. The biofilm is modelled as a 1D free boundary domain, and its evolution is governed by hyperbolic–parabolic PDEs. The initial attachment phase is modelled by considering a vanishing initial value for the free boundary. The model is investigated numerically highlighting the impact of the necrotrophic kinetic parameters, the influence of ionic strength on the initial stage of biofilm formation through bacterial attachment, and how variations in nutrient levels affect system dynamics. The numerical results demonstrate that: the ionic strength mainly governs bacterial adhesion for young biofilms and affects the biofilm ecology also in presence of a biocide; the necrotrophic metabolism of Legionella pneumophila favours its persistence in oligotrophic biofilms.

Characterization of young biofilm morphology, disinfection byproduct formation potential and toxicity of renewed water supply pipelines by phosphorus release from corroded pipes

The deterioration of drinking water quality due to corrosion of the water supply network has become inevitable and regular renewal of pipes has become a common means of doing so. Severely corroded pipes release certain nutrients (e.g., elemental phosphorus), however, little has been reported on the effect of old pipes on the young biofilm of new pipe sections and on ensuring water safety in the early stages of the water supply. The aim of our study was to model the effect of key phosphorus nutrients released from corroded old pipes on the morphological characteristics of young biofilms in new pipe sections, mediated disinfection byproducts (DBPs) production and their combined toxicity. Based on the experimental results, phosphorus showed significant differences in the morphological characteristics, spatial structure of extracellular polymers (EPS), functional abundance, disinfection byproduct formation potential (DBPsFP) and toxicity of young biofilms. Under residual chlorine (1.0 ± 0.2 mg/L) incubation, the functional abundance of young biofilm metabolism was dominant, particularly amino acid metabolism and carbohydrate metabolism. There is a dynamic balance between the trophic and shedding effects of phosphorus, where concentration changes affect young biofilm morphology and DBPFP. Relatively moderate phosphorus concentrations resulted in the highest density of PN/PS organic precursors in EPS and a clear advantage of DBPFP; relatively high phosphorus conditions had limited promotion of young biofilm, while membrane structure shedding was more pronounced, increasing young biofilm-mediated DBPs production. Nitrogen-containing disinfection byproducts (N-DBPs) in young biofilms had a clear toxicity advantage, with HANs and HNMs being key to controlling cytotoxicity and genotoxicity, respectively.

An indolizine squaraine-based water-soluble NIR dye for fluorescence imaging of multidrug-resistant bacteria and antibacterial/antibiofilm activity using the photothermal effect.

The majority of nosocomial infections are caused by bacteria with antimicrobial resistance and the formation of biofilms, such as implant-related bacterial infections and sepsis. There is an urgent need to develop new strategies for early-stage screening, destruction of multidrug-resistant bacteria, and efficient inhibition of biofilms. Organic dyes that absorb and emit in the near-infrared (NIR) region are potentially non-invasive, high-resolution, and rapid biological imaging materials. In this study, a non-toxic and biocompatible indolizine squaraine dye with water-solubilizing sulfonate groups (SO3SQ) is studied for bacterial imaging and photothermal therapy (PTT). PTT is efficient in eliminating microorganisms through local hyperthermia without the risk of developing drug-resistant bacteria. The optical properties of SO3SQ are studied extensively in phosphate-buffered saline (PBS). UV-Vis-NIR absorption spectra analysis shows a strong absorption between 650nm - 1000nm. SO3SQ allows for the wash-free fluorescence imaging of drug-resistant bacteria via NIR fluorescence imaging due to a "turn-on" fluorescence property of the dye when interacting with bacteria. Although SO3SQ exhibits no toxicity against both Gram-positive bacteria and Gram-negative bacteria, the PTT property of SO3SQ is efficient in killing bacteria as well as inhibiting and eradicating biofilms. PTT experiments demonstrate that SO3SQ reduces 90% of cell viability in bacterial strains under NIR radiation with a minimum inhibition concentration (MIC90) of >450μg/mL. The PTT property of SO3SQ can also inhibit biofilms (BIC90=1000-2000μg/mL) and eradicate both preformed young and mature biofilms (MBEC90=1500-2000μg/mL) as observed by crystal violet assays.

Efficacy and toxicity of hydrogen peroxide producing electrochemical bandages in a porcine explant biofilm model.

Effects of H2 O2 producing electrochemical-bandages (e-bandages) on methicillin-resistant Staphylococcus aureus colonization and biofilm removal were assessed using a porcine explant biofilm model. Transport of H2 O2 produced from the e-bandage into explant tissue and associated potential toxicity were evaluated. Viable prokaryotic cells from infected explants were quantified after 48 h treatment with e-bandages in three ex vivo S. aureus infection models: (1) reducing colonization, (2) removing young biofilms and (3) removing mature biofilms. H2 O2 concentration-depth profiles in explants/biofilms were measured using microelectrodes. Reductions in eukaryotic cell viability of polarized and nonpolarized noninfected explants were compared. e-Bandages effectively reduced S. aureus colonization (p =0.029) and reduced the viable prokaryotic cell concentrations of young biofilms (p =0.029) with limited effects on mature biofilms (p >0.1). H2 O2 penetrated biofilms and explants and reduced eukaryotic cell viability by 32-44% compared to nonpolarized explants. H2 O2 producing e-bandages were most active when used to reduce colonization and remove young biofilms rather than to remove mature biofilms. The described e-bandages reduced S. aureus colonization and young S. aureus biofilms in a porcine explant wound model, supporting their further development as an antibiotic-free alternative for managing biofilm infections.

Anti-Candida albicans Activity of Ononin and Other Secondary Metabolites from Platonia Insignis MART.

Candida albicans is a human pathogen that is part of the healthy microbiome. However, it is often associated with opportunistic fungal infections. The treatment of these infections is challenging because prolonged exposure to antifungal drugs can culminate in fungal resistance during therapy, and there is a limited number of available drugs. Therefore, this study investigated the antifungal activity of ononin by in silico and in vitro assays, and in Tenebrio molitor as an alternative in vivo model of infection caused by C. albicans. Ononin is an isoflavone glycoside derived from formononetin that has various biological activities. According in silico evaluation, ononin showed the best electron affinity in molecular docking with CaCYP51, with a binding free energy of -10.89 kcal/mol, superior to that of the antifungal drugs fluconazole and posaconazole. The ononin + CaCYP51 complex formed hydrogen bonds with Tyr132, Ser378, Phe380, and Met508, as well as hydrophobic connections with Tyr118, Leu121, Phe126, Leu131, Ile304, and Leu309, and interactions with the heme group. Ononin exerted anti-Candida albicans activity, with MIC between 3.9 and 7.8 µg/mL, and inhibited young and mature biofilms, with a reduction in cell density and metabolic activity of 50 to 80%. The compound was not cytotoxic to sheep red blood cells at concentrations up to 1000 µg/mL. Larvae of the mealworm T. molitor were used as an alternative in vivo model of C. albicans infection. Ononin was able to prolong larval survival at concentrations of 0.5, 1, and 5 mg/kg, and was not toxic up to a concentration of 20 mg/kg. Moreover, ononin reduced the fungal charge in treated animals. In conclusion, our results suggest that ononin has anti-Candida albicans activity and is a potential candidate for the development of new therapeutic alternatives.

Does Secondary Plant Metabolite Ursolic Acid Exhibit Antibacterial Activity against Uropathogenic Escherichia coli Living in Single- and Multispecies Biofilms?

Multispecies bacterial biofilms are the often cause of chronic recurrent urinary tract infections within the human population. Eradicating such a complex bacterial consortium with standard pharmacotherapy is often unsuccessful. Therefore, plant-derived compounds are currently being researched as an alternative strategy to antibiotic therapy for preventing bacterial biofilm formation and facilitating its eradication. Therefore, our research aimed to determine the effect of secondary plant metabolite ursolic acid (UA) on the growth and survival, the quantity of exopolysaccharides formed, metabolic activity, and morphology of uropathogenic Gram-negative rods living in single- and mixed-species biofilms at various stages of their development. Spectrophotometric methods were used for biofilm mass formation and metabolic activity determination. The survival of bacteria was established using the serial dilution assay. The decrease in survival and inhibition of biofilm creation, both single- and multispecies, as well as changes in the morphology of bacterial cells were noticed. As UA exhibited better activity against young biofilms, the use of UA-containing formulations, especially during the initial steps of urinary tract infection, seems to be reasonable. However, the future direction should be a thorough understanding of the mechanisms of UA activity as a bioactive substance.

Biofilm eradication ability of phage cocktail against Listeria monocytogenes biofilms formed on food contact materials and effect on virulence-related genes and biofilm structure

Listeria monocytogenes is a foodborne pathogen that can form biofilms in food processing facilities even under unfavorable growth environment. This study aimed to evaluate the biofilm eradication ability of Listeria-specific bacteriophage (phage) cocktail (LMPC01+02+03) against L. monocytogenes young (1 day) and mature (3 days) biofilms formed on food contact materials (FCMs: polyethylene, polypropylene, and stainless steel) at 4, 15, and 30 °C. In addition, virulence-related genes and biofilm structure parameters of the phage-treated biofilms were investigated. The biofilm eradication ability of the phage cocktail was evaluated on 96 well and MBEC plate, and the results revealed that a multiplicity-of-infection (MOI) 100 of the phage cocktail exhibited the ability of eradicate biofilms. Using MOI 100, the phage cocktail treatment on the biofilms formed on FCMs for 8 h reduced over 2 log CFU/cm2 of the young biofilms, and approximately 1 log CFU/cm2 of the mature biofilms. In addition, the phage treatment against the biofilms resulted in a significant up-regulation of two genes (flaA and motB), and up/down-regulation or no changes in three genes (hlyA, prfA, and actA). Confocal and scanning electron microscopy images revealed the loss of the biofilm matrix after the phage treatment, and quantitative analysis revealed a reduction in the structural parameters of the biofilm, except the microcolonies at the substratum level, which increased. These results suggested that MOI 100 of the phage cocktail (LMPC01+02+03) was an effective tool for eradicating L. monocytogenes biofilms formed on FCMs, and it is essential to develop a countermeasure to eradicate the biofilm remaining after phage treatment.

Anti-virulence properties of catechin-in-cyclodextrin-in-phospholipid liposome through down-regulation of gene expression in MRSA strains

Methicillin resistant Staphylococcus aureus (MRSA) is a prime pathogen responsible for various infections in human beings. Expression of virulence factors is a biggest challenge in MRSA, which results in failure of conventional antibiotic therapy. In connection to the search for natural and safe anti-virulence compounds, the present study focused to evaluate the anti-virulence potential of catechin-in-cyclodextrin-in-phospholipid liposome (CCPL) on MRSA strains. CCPL inhibited young biofilm (64.15–72.70%) as well degraded mature biofilm (55.60–63.65%) at ½ and ¼ MIC doses, which was further confirmed by scanning electron microscopy and confocal laser scanning microscope studies. CCPL was capable enough to modify the surface hydrophobicity (40.26–48.59%), reduce the EPS production (1.71–2.25 folds) and bacterial motility. In addition, CCPL inhibited the synthesis of virulence factors like slime production (0.40–0.50 folds), DNase production, hemolytic activity (28.08–49.07%), proteolytic production (14.65–18.04%), lipase production, autolysis and cell auto-aggregation. CCPL prevented the staphyloxanthin production and thereby increased the susceptibility of MRSA strains towards H2O2. Further, CCPL significantly down-regulated the virulence genes (agrA, agrC, clfA, clfB, fnbA, fnbB, icaA, icaD, hla, hld, rna III, atlA, sarA, sigB & geh). Thus, the results of present study revealed that the CCPL can effectively reduce the virulence properties and its application could inhibit the pathogenicity and also prevents the development of drug-resistance in MRSA strains.

Development of Phage Cocktails to Treat E. coli Catheter-Associated Urinary Tract Infection and Associated Biofilms.

High rates of antimicrobial resistance and formation of biofilms makes treatment of Escherichia coli catheter-associated urinary tract infections (CAUTI) particularly challenging. CAUTI affect 1 million patients per year in the United States and are associated with morbidity and mortality, particularly as an etiology for sepsis. Phage have been proposed as a potential therapeutic option. Here, we report the development of phage cocktails that lyse contemporary E. coli strains isolated from the urine of patients with spinal cord injury (SCI) and display strong biofilm-forming properties. We characterized E. coli phage against biofilms in two in vitro CAUTI models. Biofilm viability was measured by an MTT assay that determines cell metabolic activity and by quantification of colony forming units. Nine phage decreased cell viability by >80% when added individually to biofilms of two E. coli strains in human urine. A phage cocktail comprising six phage lyses 82% of the strains in our E. coli library and is highly effective against young and old biofilms and against biofilms on silicon catheter materials. Using antibiotics together with our phage cocktail prevented or decreased emergence of E. coli resistant to phage in human urine. We created an anti-biofilm phage cocktail with broad host range against E. coli strains isolated from urine. These phage cocktails may have therapeutic potential against CAUTI.

Inhibition of corrosion causing Pseudomonas aeruginosa using plasma-activated water.

The cost of Microbiologically Influenced Corrosion (MIC) significantly affects a wide range of sectors. This study aims to assess the efficiency of a novel technology based on the use of plasma-activated water (PAW) in inhibiting corrosion caused by bacteria. This study evaluated the effectiveness of PAW, produced by a plasma bubble reactor, in reducing corrosion causing Pseudomonas aeruginosa planktonic cells in tap water and biofilms were grown onto stainless steel (SS) coupons. Planktonic cells and biofilms were treated with PAW at different discharge frequencies (500-1500Hz) and exposure times (0-20min). P. aeruginosa cells in tap water were significantly reduced after treatment, with higher exposure times and discharge frequencies achieving higher reductions. Also, PAW treatment led to a gradual reduction for young and mature biofilms, achieving >4-Log reductions after 20min. Results were also used to develop two predictive inactivation models. This work presents evidence that PAW can be used to inactivate both planktonic cells and biofilms of P. aeruginosa. Experimental and theoretical results also demonstrate that reduction is dependent on discharge frequency and exposure time. This work demonstrates the potential of using PAW as means to control MIC.

Identification and characterization of Deoxyribonuclease II in planarian Dugesia japonica

Deoxyribonuclease II (DNase II) has been found to regulate inflammation, autoimmunity and apoptosis in vertebrates and invertebrates. The strong capacity of degrading DNA makes DNase II play an important role in the immune process. Planarian has become one of the model references due to its strong immune system, the environment they live makes planarians face the threat of microorganisms and injury, the strong immune system can protect planarians from the threat of bacterial and infection. In this study, we found that there was DNase in the lysis buffer of planarians, then we acquired the sequence of DjDN2s (Dugesia japonica DNase2s) and confirmed the DjDN2s were conserved DNase IIs. The predicted structure showed the active sites and binding patterns of DjDN2s. Whole-mount in situ hybridization results showed DjDN2s mainly expressed in immune organs. Quantitative real-time PCR revealed that the expression of DjDN2s upregulated in varying degrees when got hurt and challenged with bacteria, and the knockdown of DjDN2s led to the slower repair of wound. The recombinant phages which take DjDN2 also had the ability to degrade DNA and clear young biofilm of Gram-negative bacteria. Collectively, DNase II of planarian might play a role in the antimicrobial response and wound-induced response.

In vitro evaluation of the antibacterial effect of four root canal sealers on dental biofilms.

To dynamically evaluate the effect of four root canal sealers on the killing of biofilms within dentinal tubules. Dentin blocks were prepared for infection of the dentinal tubules. Enterococcus faecalis VP3-181 and multi-species bacteria from two donors were cultured. After 3days of incubation, the infected dentin specimens were rinsed with sterile water for 1min and subjected to treatment. Additionally, multi-species bacteria from donor 1 were incubated for 3weeks to allow biofilm maturation and then the specimens were subjected to treatment. Gutta-percha-treated dentin specimens comprised the control group. A root canal sealer (bioceramic sealers: EndoSequence BC Sealer, ProRoot Endo Sealer, or GuttaFlow Bioseal; and a traditional silicone-based sealer: Guttaflow 2) was spread onto the canal walls of the dentin. The specimens were examined with confocal laser scanning microscopy at 7, 30, or 60days. In the 3-day-old biofilm group, the proportion of killed bacteria decreased significantly from the first 7days of treatment to 60days of treatment for all sealers (p < 0.05). In the 3-week-old biofilm group, 60days of exposure to bioceramic sealers resulted in more significant dead bacteria than 7-day exposures of the biofilms (p < 0.05). Bioceramic sealers were more effective in killing bacteria than the GuttaFlow 2 sealer (p < 0.05). Calcium silicate-based sealers showed good antimicrobial effects against biofilms within dentinal tubules, especially in the first week in young biofilms. There is no substantive antibacterial activity observed for the examined root canal sealers against young dentinal tubule biofilms. The bioceramic root canal sealers examined demonstrate minimal additional antibacterial effects after long-term exposure to young biofilms.

Bacitracin and isothiocyanate functionalized silver nanoparticles for synergistic and broad spectrum antibacterial and antibiofilm activity with selective toxicity to bacteria over mammalian cells

Silver nanoparticles functionalized with bacitracin (BA), a cyclic peptide and isothiocyanate (ITC), a natural plant product, was fabricated. The particle size of AgNP-BA&ITC was optimized using full factorial design. The optimized particles were of 10–15 nm in size as seen under TEM and showed chemical signature of both bacitracin as well as isothiocynate in FTIR spectroscopy. XRD analysis confirmed the crystalline nature of these particles. Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) showed 21 mg/g silver content in AgNP-BA &ITC. These nanoparticles exhibited MIC in the range of 12.5–25 μg/mL and > 3 log10 reduction in cell viability for both Gram positive and Gram-negative bacteria. They clearly demonstrated biofilm inhibition (BIC90 = 150–400 μg/mL) as well as were capable of eradicating both young and mature preformed biofilms as observed by live/dead imaging and crystal violet assay. Further cytotoxicity assay suggests high selectivity (IC50/MIC90 value = 15.2–30.4) of these particles. The results in the present investigation provide role of these novel nanoparticles having substantially low silver content with reduced toxicity and good antibacterial and antibiofilm activity for external wound healing applications.

Broadcasting of amplitude- and frequency-modulated c-di-GMP signals facilitates cooperative surface commitment in bacterial lineages

Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.