Multispecies Biofilm Research Articles

Microbial composition and diversity in intraradicular biofilm formed in situ: New concepts based on next-generation sequencing.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMB) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n=10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the Microbiome Analyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

Unraveling the role of inert biomass in membrane aerated biofilm reactors for simultaneous nitrification and denitrification

This study presents an innovative 2D spatio-temporal model that sheds light on the intricate formation of biofilms, incorporating two essential biomass decay pathways: cell lysis and endogenous respiration. The model encompasses heterotrophic bacteria (HB), anaerobic heterotrophic bacteria (AHB), and autotrophic bacteria (AB), offering a comprehensive understanding of multi-species biofilm development. Through meticulous simulations, we explore the primary mechanisms behind inert biomass formation in biofilms, revealing the key roles played by the lysis of HB, AHB, and AB, as well as the endogenous respiration of HB. Moreover, the simulations reveal how species of higher abundance contribute significantly to inert biomass generation, reshaping our understanding of biofilm dynamics. Crucially, this study highlights the indispensability of considering biofilm inert biomass when modeling the nitrification and denitrification behaviors of a membrane aerated biofilm reactor (MABR). The distribution of oxygen and acetate across biofilm thickness is remarkably different when inert biomass is factored in, underscoring the necessity for a more holistic approach to modeling biofilm behavior. With the introduction of the inert biomass inclusive biofilm model, our simulations explore the interactive effects of key process conditions - bulk concentrations of oxygen ($O_{\infty}$), ammonium nitrogen ($N_{1\infty}$), acetate ($A_{\infty}$), and biofilm thickness - on the nitrification and denitrification performance of MABR. A compelling correlation emerges between higher bulk concentrations of oxygen and ammonium nitrogen and optimal nitrification rates, achieving an impressive range of 0.3 to 1.1 g ammonium/$m^{2}/d. Delving into denitrification, we observe that high $O_{\infty}$, low $N_{1\infty}$, and either high or low $A_{\infty}$ levels impede AHB formation and consequently hinder denitrification. Our findings provide a roadmap for achieving simultaneous nitrification and denitrification, contingent on specific conditions: $10[gm^{-3}] < O_{\infty} < 15[gm^{-3}]$, $12[gm^{-3}] < N_{1\infty} < 20[gm^{-3}]$, $A_{\infty}$ ranging from $3[gm^{-3}]$ to $12[gm^{-3}]$, and a biofilm thickness $> 1.4[mm]$. While our study reveals promising avenues for simultaneous nitrification and denitrification, denitrification rates still lag behind nitrification rates under the same conditions. As a result, we advocate for further investigations to devise strategies that can enhance denitrification in MABR systems. In conclusion, this study advances our knowledge of biofilm dynamics by introducing a comprehensive model and illuminating the key factors driving nitrification and denitrification performance in MABRs. These findings pave the way for improved biofilm engineering and wastewater treatment strategies, opening new horizons for sustainable environmental practices.

Antimicrobial Effect of Dental Adhesive on Cariogenic Multi-Species Biofilm

Dental caries, or tooth decay, is one of the most widespread chronic and multifactorial diseases affecting people worldwide. It is described as the localized destruction of tooth structure by acids produced from bacteria fermentation of edible carbohydrates. The disease process begins within the bacterial biofilm on the surfaces of the teeth. Streptococcus sanguinis, Sreptococcusgordonii, and Streptococcus mutans are the main organisms in the biofilm associated with health or disease conditions in the oral cavity. Streptococcus mutans is believed to be the primary cause of enamel demineralization and the development of dental caries. S. gordonii and S. sanguinis belong to a group of streptococci considered health-related commensal oral bacteria, crucial for forming a biofilm on oral hard tissues. Several types of materials with multipurpose characteristics have been incorporated into dental adhesives. Recently, the calcium salt of an acidic monomer (calcium salt of 4-methacryloxyethyl trimellitic acid, or CMET) has been included in dental adhesive and demonstrated to suppress cariogenic biofilm formation of S. mutans. However, using a single-species model may not mimic the intricate antagonistic and synergistic relations that occur in oral biofilms. Therefore, the effect on cariogenic multi-species biofilm was assessed in this study. Dental adhesive consisting of CMET and 10-methacryloyloxydecyl dihydrogen calcium phosphate (MDCP) (Bio-Coat CA) was spread over the flat-bottom surface of the 96-well plate and LED light-cured. Then it was coated with sterile saliva at 37 °C for 45 min to form an acquired pellicle for microbial attachment. The multi-species bacterial suspension containing Streptococcus mutans ATCC 25715, Streptococcus sanguinis ATCC 10556, and Streptococcus gordonii ATCC 10558 was prepared and added to the saliva-coated well. Then, the plate was incubated at 37°C in a 5% CO2 atmosphere for 24 h, 48 h, and 72 h to support the biofilm formation. The number of vital bacteria in the biofilm was determined with the WST-8 Microbial Cell Counting Kit (Dojindo Molecular Technologies, USA). All tests were done in triplicate and repeated three times. For statistical analysis, Kruskal-Wallis and Dunn’s tests were employed. The results showed that at 24 h, 48 h, and 72 h, dental adhesive with CMET could inhibit the biofilm formation of multi-species bacteria significantly compared with controls. The percentages of biofilm inhibition were 29.1%, 34.7%, and 33.2% at 24 h, 48 h, and 72 h, respectively. Dental adhesive containing CMET displayed favorable multi-species biofilm-inhibiting effects up to 72 h of biofilm growth. It is a promising adhesive for use to prevent secondary caries at the sites of restorations.

Microbial composition and diversity in intraradicular biofilm formed in situ: New concepts based on next-generation sequencing.

This study aimed to characterize the taxonomic composition of intraradicular multispecies biofilms (IMBs) formed in situ in a model to reproduce clinical conditions. Twelve palatal roots of maxillary molars had its canals prepared. Two roots were randomly selected to sterility control. Ten intraoral prosthetic appliances with lateral slots were fabricated. The roots were positioned in the slots with the canal access open to the oral cavity. Eight volunteers wore the appliance for 21 days, and two wore it at two different time points. One root from each appliance was removed and stored at -20°C until DNA extraction and sequencing (n=10). Biofilm was analyzed using next-generation sequencing and bioinformatics. The V4 hyper-variable region of the 16SrRNA gene was amplified and sequenced. For data analyses, the mothur pipeline was used for 16SrRNA processing, and subsequent analyses of the sequence dataset were performed in R using the MicrobiomeAnalyst R package. The taxonomy-based analysis of bacterial communities identified 562 operational taxonomic units (OTUs), which belonged to 93 genera, 44 families, and 8 phyla. Bacterial colonization was different for each biofilm, and samples did not have the same group of bacteria. Alpha and beta diversity analysis revealed some general patterns of sample clustering. A core microbiome of prevalent OTUs and genera was identified. IMBs were heterogeneous when analyzed individually, but some diversity patterns were found after sample clustering. The experimental model seemed to reproduce the actual biofilm composition in endodontic infections, which suggests that it may be used to evaluate disinfection protocols.

The Impact of Candida albicans in the Development, Kinetics, Structure, and Cell Viability of Biofilms on Implant Surfaces-An In Vitro Study with a Validated Multispecies Biofilm Model.

This study aimed to evaluate the impact of Candida albicans on subgingival biofilm formation on dental implant surfaces. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to compare biofilm structure and microbial biomass in the presence and absence of the fungus after periods of 24, 48, and 72 h. Quantitative polymerase chain reaction (qPCR) was used to quantify the number of viable and total micro-organisms for each of the biofilm-forming strains. A general linear model was applied to compare CLSM and qPCR results between the control and test conditions. The biofilm developed with C. albicans at 72 h had a higher bacterial biomass and a significantly higher cell viability (p < 0.05). After both 48 and 72 h of incubation, in the presence of C. albicans, there was a significant increase in counts of Fusobacterium nucleatum and Porphyromonas gingivalis and in the cell viability of Streptococcus oralis, Aggregatibacter actinomycetemcomitans, F. nucleatum, and P. gingivalis. Using a dynamic in vitro multispecies biofilm model, C. albicans exacerbated the development of the biofilm grown on dental implant surfaces, significantly increasing the number and cell viability of periodontal bacteria.

Chestnut Honey Is Effective against Mixed Biofilms at Different Stages of Maturity.

The irresponsible overuse of antibiotics has increased the occurrence of resistant bacterial strains, which represents one of the biggest patient safety risks today. Due to antibiotic resistance and biofilm formation in bacteria, it is becoming increasingly difficult to suppress the bacterial strains responsible for various chronic infections. Honey was proven to inhibit bacterial growth and biofilm development, offering an alternative solution in the treatment of resistant infections and chronic wounds. Our studies included chestnut honey, valued for its high antibacterial activity, and the bacteria Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and S. epidermidis, known to form multi-species biofilm communities. Minimum inhibitory concentrations (MIC) of chestnut honey were determined for each bacterial strain. Afterwards, the mixed bacterial biofilms were treated with chestnut honey at different stages of maturity (incubation times: 2, 4, 6, 12, 24 h). The extent of biofilm inhibition was measured with a crystal violet assay and demonstrated by scanning electron microscopy (SEM). As the incubation time increased and the biofilm became more mature, inhibition rates decreased gradually. The most sensitive biofilm was the combination MRSA-S. epidermidis, with a 93.5% inhibition rate after 2 h of incubation. Our results revealed that chestnut honey is suitable for suppressing the initial and moderately mature stages of mixed biofilms.

Antifouling activity of aquatic macrophyte extracts on estuarine bacterial biofilms

ABSTRACT Ecologically safe antifouling solutions have received growing attention since the acknowledgment of the environmental risks of traditional biocides used currently. Given that bacteria constitute the initial stage of the biofouling process, this work aims to evaluate the antifouling potential of aquatic macrophyte extracts in inhibiting biofilm-forming bacteria and to evaluate their ecotoxicological effects on target and nontarget organisms (Thalassiosira pseudonana and Nitokra sp.). For this, the ability of 25 macrophyte extracts to inhibit and eradicate single and multispecies estuarine biofilms was evaluated. The extracts and respective serial dilutions that showed a biofilm inhibitory effect ≥ 60% were evaluated in ecotoxicological assays. Of the 25 extracts, only 6 extracts inhibited the biofilm ≥ 60%. The extracts of Cabomba caroliniana and Schoenoplectus californicus stood out as having a biofilm inhibitory effect ≥ 80% (p < 0.05) for bacterial isolates and ≥ 70% (p < 0.05) for multispecies biofilms. Furthermore, these extracts showed no toxic effects on nontarget organisms. These results demonstrate the biotechnological potential of macrophyte compounds.

Autoinducer-2 produced by oral microbial flora and alveolar bone loss in periodontitis.

The aim of this study was to investigate the association between autoinducer-2 (AI-2) of oral microbial flora and the alveolar bone destruction in periodontitis to determine if AI-2 may have the potential that monitor periodontitis and predict bone loss. Plaque biofilm was the initiating factor of periodontitis and the essential factor of periodontal tissue destruction. The formation of biofilms depended on the complex regulation of the quorum sensing (QS) system, in which bacteria could sense changes in surrounding bacterial density by secreting the autoinducer (AI) to regulate the corresponding physiological function. Most oral bacteria also communicated with each other to form biofilms administrating the QS system, which implied that the QS system of periodontal pathogens was related to periodontitis, but the specific relationship was unknown. We collected the gingival crevicular fluid (GCF) samples and measured the concentration of AI-2 in samples using the Vibrio harveyi BB180 bioluminescent-reporter system. To explore the interaction between AI-2 and bone metabolism, we utilized AI-2 purified from Fusobacterium nucleatum to investigate the impact of F. nucleatum AI-2 on osteoclast differentiation. Moreover, we constructed murine periodontitis models and multi-species biofilm models to study the association between AI-2 and periodontal disease progression. The AI-2 concentration in GCF samples increased along with periodontal disease progression (p < .0001). F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner. In the periodontitis mice model, the CEJ-ABC distance in the F. nucleatum AI-2 treatment group was higher than that in the simple ligation group (p < .01), and the maxilla of the mice in the group exhibited significantly lower BMD and BV/TV values (p < .05). We demonstrated that the AI-2 concentration varied with the alveolar bone destruction in periodontitis, and it may have the potential for screening periodontitis. F. nucleatum AI-2 promoted osteoclast differentiation in a dose-dependent manner and aggravated bone loss.

In vitro efficacy of Er:YAG laser-activated irrigation versus passive ultrasonic irrigation and sonic-powered irrigation for treating multispecies biofilms in artificial grooves and dentinal tubules: an SEM and CLSM study

BackgroundMultispecies biofilms located in the anatomical intricacies of the root canal system remain the greatest challenge in root canal disinfection. The efficacy of Er:YAG laser-activated irrigation techniques for treating multispecies biofilms in these hard-to-reach areas has not been proved. The objective of this laboratory study was to evaluate the effectiveness of two Er:YAG laser-activated irrigation techniques, namely, photon-induced photoacoustic streaming (PIPS) and shock wave-enhanced emission photoacoustic streaming (SWEEPS), in treating multispecies biofilms within apical artificial grooves and dentinal tubules, in comparison with conventional needle irrigation (CNI), passive ultrasonic irrigation (PUI), and sonic-powered irrigation (EDDY). Two types of multispecies root canal biofilm models were established in combination with two assessment methods using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) with the aim to obtain more meaningful results.MethodsNinety extracted human single-rooted premolars were chosen for two multispecies biofilm models. Each tooth was longitudinally split into two halves. In the first model, a deep narrow groove was created in the apical segment of the canal wall. After cultivating a mixed bacterial biofilm for 4 weeks, the split halves were reassembled and subjected to five irrigation techniques: CNI, PUI, EDD, PIPS, and SWEEPS. The residual biofilms inside and outside the groove in Model 1 were analyzed using SEM. For Model 2, the specimens were split longitudinally once more to evaluate the percentage of killed bacteria in the dentinal tubules across different canal sections (apical, middle, and coronal thirds) using CLSM. One-way analysis of variance and post hoc multiple comparisons were used to assess the antibiofilm efficacy of the 5 irrigation techniques.ResultsRobust biofilm growth was observed in all negative controls after 4 weeks. In Model 1, within each group, significantly fewer bacteria remained outside the groove than inside the groove (P < 0.05). SWEEPS, PIPS and EDDY had significantly greater biofilm removal efficacy than CNI and PUI, both from the outside and inside the groove (P < 0.05). Although SWEEPS was more effective than both PIPS and EDDY at removing biofilms inside the groove (P < 0.05), there were no significant differences among these methods outside the groove (P > 0.05). In Model 2, SWEEPS and EDDY exhibited superior bacterial killing efficacy within the dentinal tubules, followed by PIPS, PUI, and CNI (P < 0.05).ConclusionEr:YAG laser-activated irrigation techniques, along with EDDY, demonstrated significant antibiofilm efficacy in apical artificial grooves and dentinal tubules, areas that are typically challenging to access.

Effect of supplementary sodium hypochlorite agitation techniques on an exvivo oral multispecies biofilm during passive disinfection of simulated immature roots.

To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an exvivo oral multispecies biofilm during passive disinfection of simulated immature roots. Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova. 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples (p < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups (p < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium, Actinomyces, Porphyromonas, and Capnocytophaga. The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.

Informed development of a multi-species biofilm in chronic obstructive pulmonary disease.

Recent evidence indicates that microbial biofilm aggregates inhabit the lungs of COPD patients and actively contribute towards chronic colonization and repeat infections. However, there are no contextually relevant complex biofilm models for COPD research. In this study, a meta-analysis of the lung microbiome in COPD was used to inform development of an optimized biofilm model composed of genera highly associated with COPD. Bioinformatic analysis showed that although diversity matrices of COPD microbiomes were similar to healthy controls, and internal compositions made it possible to accurately differentiate between these cohorts (AUC = 0.939). Genera that best defined these patients included Haemophilus, Moraxella and Streptococcus. Many studies fail to account for fungi; therefore, Candida albicans was included in the creation of an interkingdom biofilm model. These organisms formed a biofilm capable of tolerating high concentrations of antimicrobial therapies with no significant reductions in viability. However, combined therapies of antibiotics and an antifungal resulted in significant reductions in viable cells throughout the biofilm (p < 0.05). This biofilm model is representative of the COPD lung microbiome and results from in vitro antimicrobial challenge experiments indicate that targeting both bacteria and fungi in these interkingdom communities will be required for more positive clinical outcomes.

Inhibition of Biofilm Formation in Cutibacterium acnes, Staphylococcus aureus, and Candida albicans by the Phytopigment Shikonin.

Skin microbiota, such as acne-related Cutibacterium acnes, Staphylococcus aureus, and fungal Candida albicans, can form polymicrobial biofilms with greater antimicrobial tolerance to traditional antimicrobial agents and host immune systems. In this study, the phytopigment shikonin was investigated against single-species and multispecies biofilms under aerobic and anaerobic conditions. Minimum inhibitory concentrations of shikonin were 10 µg/mL against C. acnes, S. aureus, and C. albicans, and at 1-5 µg/mL, shikonin efficiently inhibited single biofilm formation and multispecies biofilm development by these three microbes. Shikonin increased porphyrin production in C. acnes, inhibited cell aggregation and hyphal formation by C. albicans, decreased lipase production, and increased hydrophilicity in S. aureus. In addition, shikonin at 5 or 10 µg/mL repressed the transcription of various biofilm-related genes and virulence-related genes in C. acnes and downregulated the gene expression levels of the quorum-sensing agrA and RNAIII, α-hemolysin hla, and nuclease nuc1 in S. aureus, supporting biofilm inhibition. In addition, shikonin prevented multispecies biofilm development on porcine skin, and the antimicrobial efficacy of shikonin was recapitulated in a mouse infection model, in which it promoted skin regeneration. The study shows that shikonin inhibits multispecies biofilm development by acne-related skin microbes and might be useful for controlling bacterial infections.

Type VI secretion system drives bacterial diversity and functions in multispecies biofilms

Type VI secretion system (T6SS) plays an essential role in interspecies interactions and provides an advantage for a strain with T6SS in multispecies biofilms. However, how T6SS drives the bacterial community structure and functions in multispecies biofilms still needs to be determined. Using gene deletion and Illumina sequencing technique, we estimated bacterial community responses in multispecies biofilms to T6SS by introducing T6SS-containing Pseudomonas putida KT2440. Results showed that the niche structure shifts of multispecies biofilms were remarkably higher in the presence of T6SS than in the absence of T6SS. The presence of T6SS significantly drove the variation in microbial composition, reduced the alpha-diversity of bacterial communities in multispecies biofilms, and separately decreased and increased the relative abundance of Proteobacteria and Bacteroidota. Co-occurrence network analysis with inferred putative bacterial interactions indicated that P. putida KT2440 mainly displayed strong negative associations with the genera of Psychrobacter, Cellvibrio, Stenotrophomonas, and Brevundimonas. Moreover, the function redundancy index of the bacterial community was strikingly higher in the presence of T6SS than in the absence of T6SS, regardless of whether relative abundances of bacterial taxa were inhibited or promoted. Remarkably, the increased metabolic network similarity with T6SS-containing P. putida KT2440 could enhance the antibacterial activity of P. putida KT2440 on other bacterial taxa. Our findings extend knowledge of microbial adaptation strategies to potential bacterial weapons and could contribute to predicting biodiversity loss and change in ecological functions caused by T6SS.

Ultraviolet C as a method of disinfecting medical silicone used in facial prostheses: An in vitro study – Part 2

Statement of problemDisinfection is an important factor in preserving facial prostheses and maintaining tissue health. However, whether disinfection with ultraviolet C is an effective disinfection method is unclear. PurposeThe purpose of this in vitro study was to evaluate the effectiveness of irradiation with different exposure durations of an ultraviolet-C light-emitting diode in the disinfection of the silicone (A-588–1; Factor II) used in facial prostheses. Material and methodsA total of 216 specimens were prepared, contaminated by multispecies biofilm, and divided into 9 groups (n=24) for different treatments: chlorhexidine 0.12% (G CHG), ultraviolet-C light-emitting diode for 5 minutes (G UVC5), ultraviolet-C light-emitting diode for 10 minutes (G UVC10), ultraviolet-C light-emitting diode for 20 minutes (G UVC20), their respective untreated controls (Gcontrol CHG, Gcontrol UVC5, Gcontrol UVC10, Gcontrol UVC20), and dimethyl sulfoxide (G DMSO) as the negative control. Cell viability was measured by using the methyl tetrazolium salt (MTT) method. Two statistical analyses were performed. First, a 2×3 ANOVA was carried out to compare the control groups (Gcontrol UVC5, Gcontrol UVC10, and Gcontrol UVC20) and the experimental groups of UV-C LED light with different exposure durations (G UVC5, G UVC10, and G UVC20). The second analysis was performed using generalized linear models to compare the optical density of the groups (G UVC5, G UVC10, G UVC20, G CHG, and G DMSO). ResultsCell viability results demonstrated a microbial reduction after exposure to the ultraviolet-C light-emitting diode for 20 minutes (G UVC20) compared with untreated controls (P<.05). The 5- and 10-minute exposures were statistically similar to their respective control groups (P>.05). The 20 minutes exposure had the lowest average optical density value, being statistically different from the 5-minute exposure (P<.05). A 20-minute exposure to the ultraviolet-C light-emitting diode (G UVC20) was similarly effective when compared with the standard disinfection treatment (G CHG) and dimethyl sulfoxide (G DMSO) (P>.05). ConclusionsIrradiation with an ultraviolet-C light-emitting diode for 20 minutes decreased the in vitro microbial cell viability on the medical silicone used in facial prostheses.

Ciprofloxacin Poly(β-amino ester) Conjugates Enhance Antibiofilm Activity and Slow the Development of Resistance.

To tackle the emerging antibiotic resistance crisis, novel antimicrobial approaches are urgently needed. Bacterial biofilms are a particular concern in this context as they are responsible for over 80% of bacterial infections and are inherently more recalcitrant toward antimicrobial treatments. The high tolerance of biofilms to conventional antibiotics has been attributed to several factors, including reduced drug diffusion through the dense exopolymeric matrix and the upregulation of antimicrobial resistance machinery with successful biofilm eradication requiring prolonged high doses of multidrug treatments. A promising approach to tackle bacterial infections involves the use of polymer drug conjugates, shown to improve upon free drug toxicity and bioavailability, enhance drug penetration through the thick biofilm matrix, and evade common resistance mechanisms. In the following study, we conjugated the antibiotic ciprofloxacin (CIP) to a small library of biodegradable and biocompatible poly(β-amino ester) (PBAE) polymers with varying central amine functionality. The suitability of the polymers as antibiotic conjugates was then verified in a series of assays including testing of efficacy and resistance response in planktonic Gram-positive and Gram-negative bacteria and the reduction of viability in mono- and multispecies biofilm models. The most active polymer within the prepared PBAE-CIP library was shown to achieve an over 2-fold increase in the reduction of biofilm viability in a Pseudomonas aeruginosa monospecies biofilm and superior elimination of all the species present within the multispecies biofilm model. Hence, we demonstrate that CIP conjugation to PBAEs can be employed to achieve improved antibiotic efficacy against clinically relevant biofilm models.

Electrochemical Characterization of Two Gut Microbial Strains Cooperatively Promoting Multiple Sclerosis Pathogenesis.

In this study, we explored the extracellular electron transfer (EET) capabilities of two bacterial strains, OTU0001 and OTU0002, which are demonstrated in biofilm formation in mouse gut and the induction of autoimmune diseases like multiple sclerosis. OTU0002 displayed significant electrogenic behaviour, producing microbial current on an indium tin-doped oxide electrode surface, particularly in the presence of glucose, with a current density of 60 nA/cm2. The presence of cell-surface redox substrate potentially mediating EET was revealed by the redox-based staining method and electrochemical voltammetry assay. However, medium swapping analyses and the addition of flavins, a model redox mediator, suggest that the current production is dominated by soluble endogenous redox substrates in OTU0002. Given redox substrates were detected at the cell surface, the secreted redox molecule may interact with the cellular surface of OTU0002. In contrast to OTU0002, OTU0001 did not exhibit notable electrochemical activity, lacking cell-surface redox molecules. Further, the mixture of the two strains did not increase the current production from OTU0001, suggesting that OTU0001 does not support the EET mechanism of OTU0002. The present work revealed the coexistence of EET and non-EET capable pathogens in multi-species biofilm.

Alginate Gel Encapsulated with Enzybiotics Cocktail Is Effective against Multispecies Biofilms.

The development of new and effective antibacterials for pharmaceutical or cosmetic skin care that have a low potential for the emergence and expansion of bacterial resistance is of high demand in scientific and applied research. Great hopes are placed on alternative agents such as bactericidal peptidoglycan hydrolases, depolymerases, etc. Enzybiotic-based preparations are being studied for the treatment of various infections and, among others, can be used as topical formulations and dressings with protein-polysaccharide complexes. Here, we investigate the antibiofilm properties of a novel enzybiotic cocktail of phage endolysin LysSi3 and bacteriocin lysostaphin, formulated in the alginate gel matrix and its ability to control the opportunistic skin-colonizing bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, as well as mixed-species biofilms. Our results propose that the application of SiL-gel affects different components of biofilm extracellular polymeric substances, disrupts the matrix, and eliminates the bacteria embedded in it. This composition is highly effective against biofilms composed of Gram-negative and Gram-positive species and does not possess significant cytotoxic effects. Our data form the basis for the development of antibacterial skin care products with a gentle but effective mode of action.

Evaluation of approaches to increase the effectiveness of various disinfectants against biofilm communities of different ages

Disinfectants are used as the main agents against microorganisms circulating on the surfaces of food enterprises. However, the adaptive ability of microorganisms to form biofilms complicates the process of surface cleaning and reduces the effectiveness of disinfectants. Modern disinfectants act against freely circulating microflora, but it is known that they are not always effective against biofilms. The purpose of this study was to investigate effective disinfectant compositions with bactericidal effect on binary bacterial biofilms of different ages. The article describes the effects of disinfectants based on chlorine, peracetic acid and quaternary ammonium compounds with enzymes in concentrations recommended by the manufacturer and increased several times on Salmonella 38, Brochothrix thermosphacta 2726 and Staphylococcus equorum 2736 planktonic cultures and binary biofilms. Binary biofilms of different ages (2 and 9 days old) were exposed to disinfectants with various active ingredients in combination with adjuvants, i. e. hydrogen peroxide 6% and various concentrations of isopropyl alcohol (30%). All products in concentrations recommended by the manufacturer did not have a disinfectant effect against the studied biofilm cultures. As a result of the work, it was found that the most effective disinfectants against multispecies biofilms were quaternary ammonium compounds in combination with enzymes and chlorine in combination with isopropyl alcohol (30%). The results obtained allow to expand knowledge about effective methods for controlling biofilms.

Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Inhibitory effect of Streptococcus mutans antisense vicK RNA regulating the cariogenicity of oral streptococci multi-species biofilm

Objective: To investigate the regulative effects of Streptococcus mutans (Sm) antisense vicK RNA (ASvicK) on the multi-species biofilm formed by three common oral streptococci (Sm, Streptococcus sanguinis, and Streptococcus gordonii) (Sm+Ss+Sg). Methods: ASvicK over-expression strain was constructed by using a recombinant plasmid, and three-species biofilm UA159+Ss+Sg and ASvicK+Ss+Sg were cultured. The phenotypes of biofilms were detected by scanning electron microscopy (SEM). Crystal violet (CV) assay was used to detect biofilm biomass. Lactate kit and anthrone-sulfuric acid colorimetric assay were used to determine the abilities of lactic acid and exopolysaccharides production, respectively. The proportions of three-species and expression levels of the cariogenic-related genes in biofilms were detected by TaqMan fluorescence quantitative PCR and real-time fluorescence quantitative PCR. A biofilm demineralization model of human enamel slabs was further constructed, and the hardness of enamel surface was detected. Results: Compared to UA159+Ss+Sg, over-expression of ASvicK could inhibit biofilm formation and lactic acid production in ASvicK+Ss+Sg biofilm significantly decreased by 78.93% (P<0.001) and 62.23% (P<0.001), respectively. With ASvicK over-expression, the amounts of water-insoluble and-soluble glucoses in ASvicK+Ss+Sg biofilm were reduced respectively by 39.13% (P<0.001) and 68.00% (P<0.001). Compared to the UA159+Ss+Sg Group, the proportion of Sm, the cariogenic bacteria, showed 33.00% reduction (P<0.01) in Sm+Ss+Sg biofilm, and the gene expressions of cariogenic-relative genes vicK/X, gtfB/C/D, and ftf significantly decreased (P<0.05). The micro-hardness value of enamel slabs after demineralization by ASvicK+Ss+Sg biofilm was significantly increased to 183.84% (P<0.001). Conclusions: ASvicK over-expression could reduce the Sm proportion and weaken the cariogenicity of oral Streptococcus biofilm, thereby possibly slowing down the progression of caries.