Biofilm Bacteria Research Articles

Transcriptomics analysis of the mechanism behind Lactobacillus rhamnosus Gr18 biofilm formation in an Mn2+-deficient environment

Stress environments promote lactic acid bacteria (LAB) biofilm formation. Therefore, this study assessed the ability of Lactobacillus rhamnosus (L. rhamnosus) Gr18 to form biofilm in stress conditions. An Mn2+-deficient environment exhibited the most significant biofilm content increase of 27.9% compared to the normal conditions. Two key phases were selected to elucidate the regulatory mechanisms behind biofilm formation in Mn2+-deficient environments. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyzes, identified 368 significantly up-regulated and 413 substantially down-regulated genes during the attachment phase, suggesting that L. rhamnosus Gr18 facilitated the transition from the planktonic to the biofilm state by increasing EPS and lipid synthesis via the modulation of the LuxS/AI-2 and, SHP/Rgg pathway, fatty acid biosynthesis, ABC transporters and phosphatase system (PTS). Furthermore, 441 up-regulated genes and 162 down-regulated genes displayed significant changes during the maturation phase. LuxS/AI-2, ABC transporters, PTS, and NprX-NprR regulated lipopeptide production, which increased the extracellular matrix content and intercellular adhesion, leading to biofilm maturation. Therefore, L. rhamnosus Gr18 promoted biofilm formation by altering the extracellular matrix content, adhesion, and motility in Mn2+-deficient environments. This study provided a theoretical basis for improving LAB utilization for commercial production.

Evaluation of the relationship between periodontal diseases and oxidative stress parameters in cats

Periodontal disease, which is defined as inflammation of the tissues and supporting structures surrounding the teeth, can be observed in cats starting from the age of 2. Periodontal diseases start with Gingivitis, which is the early stage of periodontal disease. Gingivitis can arise from inflammation of the gums due to plaque, a white or yellowish biofilm of bacteria on the tooth surface, and the toxins produced by these bacteria. It can also result from inflammation of the periodontal tissues, including dental calculus or other periodontal tissues. A total of 242 cats were brought to the clinic for surgical diseases and 14 cats (5.78%) were found to have periodontal diseases. Although there have been several studies on oxidative stress, there are very few publications investigating the relationship between oxidative stress and periodontal diseases in cats. The aim of this study is to measure serum MDA, IMA and GSH concentrations and SOD and CAT activities in cats with periodontal diseases and to evaluate the relationship between oxidant and antioxidant status, which are indicators of oxidative stress. In the study, blood samples taken from cats with periodontal disease were centrifuged and serum was removed. MDA and IMA levels of cats with periodontal disease were found to be significantly higher than in cats that recovered after treatment (P<0.001). SOD, CAT and GSH levels were determined to be significantly lower cats with periodontal disease than recovered cats (P<0.001). In conclusion, this study reveals that there is a relationship between periodontal diseases and oxidant/antioxidant balance in cats and it shows that the oxidative stress develop due to the increase of free radicals.

Lysine aggregates‐based nanostructured antimicrobial peptides for cariogenic biofilm microenvironment‐activated caries treatment

AbstractDental caries is one of the most prevalent and costly biofilm‐induced oral diseases that causes the deterioration of the mineralized tooth tissue. Traditional antimicrobial agents like antibiotics and antimicrobial peptides (AMPs) struggle to effectively eradicate bacteria in biofilms without eliciting resistance. Herein, we demonstrate the construction of FeOOH@Fe‐Lysine@Au nanostructured AMPs (nAMPs) distinguished by their AMP‐like antibacterial activity and self‐producing reactive oxygen species (ROS) capacity for caries treatment. On the one hand, FeOOH@Fe‐Lysine@Au nAMPs can catalyze glucose oxidation to generate ROS within the cariogenic biofilm microenvironment, resulting in the disintegration of the extracellular polymeric substance matrix and the exposure of bacteria. On the other hand, FeOOH@Fe‐Lysine@Au nAMPs can attach to bacterial surfaces via electrostatic attractions, proceeding to damage membranes, disrupt metabolic pathways, and inhibit protein synthesis through the aggregated lysine and the generated ROS. Based on this antibacterial mechanism, FeOOH@Fe‐Lysine@Au nAMPs can effectively eradicate Streptococcus mutans and its associated biofilm, significantly impeding the progression of dental caries. Given the straightforward and cost‐efficient preparation of FeOOH@Fe‐Lysine@Au nAMPs compared to AMPs that require specific sequences, and their minimal adverse impacts on gingival/palatal tissues, major organs, and oral/gut microbiomes, our research may promote the development of novel therapeutic agents in dental health maintenance.

Nonlinear responses of biofilm bacteria to alkyl-chain length of parabens by DFT calculation

Parabens can particularly raise significant concerns regarding the disruption of microbial ecology due to their antimicrobial properties. However, the responses of biofilm bacteria to diverse parabens with different alkyl-chain length remains unclear. Here, theoretical calculations and bioinformatic analysis were performed to decipher the influence of parabens varying alkyl-chain lengths on the biofilm bacteria. Our results showed that the disturbances in bacterial community did not linearly response to the alkyl-chain length of parabens, and propylparaben (PrP), with median chain length, had more severe impact on bacterial community. Despite the fact that paraben lethality linearly increased with chain length, the PrP had a higher chemical reactions potential than parabens with shorter or longer alkyl-chain. The chemical reactions potential was critical in the nonlinear responses of bacterial community to alkyl-chain length of parabens. PrP could impose selective pressure to disturb the bacterial community, because it had a more profound contribution to deterministic assembly process. Furthermore, N-acyl-homoserine lactones was also significantly promoted under PrP exposure, confirming that PrP could affect the bacterial community by influencing the quorum-sensing system. Overall, our study reveals the nonlinear responses of bacterial communities to the alkyl-chain lengths of parabens and provides insightful perspectives for the better regulation of parabens. Environmental ImplicationParabens are recognized as emerging organic pollutants, which specially raise great concerns due to their antimicrobial properties disturbing microbial ecology. However, few study have addressed the relationship between bacterial community responses and the molecular structural features of parabens with different alkyl-chain length. This investigation revealed nonlinear responses of the bacterial community to the alkyl-chain length of parabens through DFT calculation and bioinformatic analysis and identified the critical roles of chemical reactions potential in nonlinear responses of bacterial community. Our results benefit the precise evaluation of ecological hazards posed by parabens and provide useful insights for better regulation of parabens.

Supragingival dental biofilm profile and biofilm control during orthodontic treatment with fixed orthodontic appliance: A randomized controlled trial

ObjectiveThe effectiveness of supragingival dental biofilm control during orthodontic treatment and changes in the bacterial profile were analyzed. DesignSixty-four participants aged 12–22 years (57% female) were included in the study. Participants underwent orthodontic treatment with fixed appliances and were randomly assigned to one of the three groups, which during a period of one month: (I) used chlorhexidine digluconate (CHX), (II) used high concentration of fluoride (F) gel and (III) performed standard oral hygiene. The plaque and gingivitis index, pH of biofilm and white spot lesions (WSL) were assessed. Changes of the bacteria in the biofilm were analyzed by the quantitative polymerase chain reaction ResultsIncrease in the plaque index, pH of biofilm, and WSL was observed during orthodontic treatment with standard oral hygiene. Large interindividual variability was present, and the effects of one-month use of fluorides and CHX on clinical parameters were not significant. Despite standard hygiene the abundance of studied biofilm bacteria increased - the most Streptoccocus mutans (14.2x) and S. salivarius (3.3x), moderate Veillonella parvula (3x) and the least S. sobrinus (2.3x) and Agregatibacter actinomycetemcomitans (1.9x). The use of CHX reduced S. sobrinus (2.2x) and A. actinomycetemcomitans (1.9x). Fluoride use reduced A. actinomycetemcomitans (1.3x) and S. sobrinus (1.2x). Fluorides better controlled S. mutans than CHX. ConclusionBacterial biomass in supragingival biofilm increased during treatment with metal orthodontic appliances, with greater increase in cariogenic bacteria than periopathogens. Fluoride controlled S. mutans, while CHX S. sobrinus and A. actinomycetemcomitans.

Chain elongation in continuous microbial electrosynthesis cells: The effect of pH and precursors supply

Microbial electrosynthesis (MES) enables the production of carbon-neutral chemicals using CO2 as a carbon source. Acetic acid is the main MES product, but recent studies show the direct production of elongated carboxylic acids, e.g., butyric and caproic acid. However, the production of elongated acids in MES systems is still inefficient due to the low growth rates of acetogenic bacteria and to limited solventogenic rates. Subsequently, researchers have produced elongated carboxylic acids directly from acetic acid or have operated MES systems at low pH to favor solventogenesis. However, the effect the addition of different chain elongation precursors and the operation pH exerts in the bioelectrochemical production of elongated acids remains unclear. To investigate this, three pH-controlled MES systems were operated in this study with continuous liquid and gas supply. MES systems elongating acetic acid at pH 6 achieved higher butyric (0.71 vs. 0.42 g L−1) and caproic acid (0.71 vs. 0.42 g L−1) titers in the absence of CO2 sparging. Additionally, lowering the pH to 5 in the MES systems fed with CO2 and acetic acid improved the elongated acids titers, reaching 0.72 g L−1 butyric and 0.33 g L−1 caproic acid. The 16 S rRNA analysis showed the community was dominated by Oscillibacter at pH 6, and by Clostridium at pH 5. Furthermore, the first scanning electron microscopy pictures revealing biofilm stratification in MES cathodes were taken in this study, where homogeneous rod-shaped bacteria biofilm layers, in contact with the graphite cathode, were covered by heterogeneous biofilm layers.

Mussel-inspired interface deposition strategy for mesoporous metal-phenolic nanospheres with superior antioxidative, photothermal and antibacterial performance

Metal-phenolic networks (MPNs) have emerged as a versatile and multifunctional platform applied in bioimaging, disease treatment, electrocatalysis, and water purification. The synthesis of MPNs with mesoporous frameworks and ultra-small diameters (<200 nm), crucial for post-modification, cargo loading, and mass transport, remains a formidable challenge. Inspired by mussel chemistry, mesoporous metal-phenolic nanospheres (MMPNs) are facilely prepared by direct deposition of the metal-polyphenol complex on the interface of oil nano-droplets composed of block copolymers/1,3,5-trimethylbenzene followed by a spontaneous template-removal process. Due to the penetrable and stable networks, the oil nano-droplets gradually leak from the networks driven by shear stress during the stirring process. As a result, MMPNs are obtained without additional template removal procedures such as solvent extraction or high-temperature calcination. The materials have a large pore size (∼12.1 nm), uniform spherical morphology with a small particle size (∼99 nm), and a large specific surface area (49.8 m2 g−1). Due to the abundant phenolic hydroxyl groups, the MMPNs show excellent antioxidative property. The MMPNs also have excellent photothermal property, whose photothermal conversion efficiency was 40.9 %. Moreover, the phenolic hydroxyl groups can reduce Ag+ in situ to prepare Ag nanoparticles loaded MMPNs composites, which have excellent inhibition performance of drug-resistant bacteria biofilm.

Design and evaluation of an MMP-9-responsive hydrogel for vital pulp therapy

ObjectiveTo design and evaluate a matrix metalloproteinase 9 (MMP-9)-responsive hydrogel for vital pulp therapy. MethodsA peptide linker with optimized sensitivity toward MMP-9 was crosslinked with 4-arm poly (ethylene glycol)-norbornene (PEG-NB) by thiol-norbornene photo-polymerization. This resulted in the formation of a hydrogel network in which the peptide IDR-1002 was incorporated. Hydrogel characterization and gelation kinetics were examined with Fourier-transform infrared spectroscopy, scanning electron microscopy, rheological testing, and swelling evaluation. Hydrogel degradation was examined through multiple exposure to pre-activated MMP-9, to simulate flare-ups of dental pulp inflammation. The IDR-1002 released from degraded hydrogels was measured with high-performance liquid chromatography. Effect of IDR-1002 released from hydrogels on one-week-old multispecies oral biofilms was evaluated using confocal laser scanning microscopy. ResultsMMP-9-responsive, injectable, and photo-crosslinkable hydrogels were successfully synthesized. When hydrogel degradation and release of IDR-1002 were examined with exposure to pre-activated MMP-9, IDR-1002 release was significantly correlated with elevated levels of MMP-9 (p < 0.05). The effectiveness of IDR-1002 in killing bacteria in multispecies oral biofilms was significantly enhanced when the hydrogels were immersed in 10 nM or 20 nM pre-activated MMP-9, compared to immersion in phosphate-buffered saline (p < 0.05). ConclusionsThe MMP-9-responsive hydrogel is a promising candidate for on-demand delivery of bioactive agent in vital pulp therapy. Clinical significanceMMP-9 is one of the most important diagnostic and prognostic biomarkers for pulpitis. An MMP-9-responsive hydrogel has potential to be used as an in-situ on-demand release system for the diagnosis and treatment of dental pulp inflammation.

Comparisons of Numerical and Solitary Wave Solutions for the Stochastic Reaction–Diffusion Biofilm Model including Quorum Sensing

This study deals with a stochastic reaction–diffusion biofilm model under quorum sensing. Quorum sensing is a process of communication between cells that permits bacterial communication about cell density and alterations in gene expression. This model produces two results: the bacterial concentration, which over time demonstrates the development and decomposition of the biofilm, and the biofilm bacteria collaboration, which demonstrates the potency of resistance and defense against environmental stimuli. In this study, we investigate numerical solutions and exact solitary wave solutions with the presence of randomness. The finite difference scheme is proposed for the sake of numerical solutions while the generalized Riccati equation mapping method is applied to construct exact solitary wave solutions. The numerical scheme is analyzed by checking consistency and stability. The consistency of the scheme is gained under the mean square sense while the stability condition is gained by the help of the Von Neumann criteria. Exact stochastic solitary wave solutions are constructed in the form of hyperbolic, trigonometric, and rational forms. Some solutions are plots in 3D and 2D form to show dark, bright and solitary wave solutions and the effects of noise as well. Mainly, the numerical results are compared with the exact solitary wave solutions with the help of unique physical problems. The comparison plots are dispatched in three dimensions and line representations as well as by selecting different values of parameters.

Selective antibacterial and antibiofilm activity of chlorinated hemicyanine against gram-positive bacteria

Antibiotic-free therapies are highly needed due to the limited success of conventional approaches especially against biofilm related infections. In this direction, antimicrobial phototherapy, either in the form of antimicrobial photothermal therapy (aPTT) or antimicrobial photodynamic therapy (aPDT), have appeared to be highly promising candidates in recent years. These are local and promising approaches for antibiotic resistant bacterial infections and biofilms. Organic small photosensitizers (PSs) are extensively preferred in antimicrobial phototherapy applications as they offer a great opportunity to combine therapeutic action (aPTT, aPDT or both) with fluorescence imaging on a single molecule. In this study, the bactericidal effect of cationic chlorinated hemicyanine (Cl-Hem)-based type I PS, which can function as a dual aPDT/aPTT agent, was investigated on both planktonic cells and biofilms of different gram-positive (E. faecalis and S. epidermidis) and gram-negative bacteria (P. aeruginosa and K. pneumoniae) with and without 640 nm laser irradiation. Cl-Hem was shown to induce a selective phototheranostic activity against gram-positive bacteria (E. faecalis and S. epidermidis). Cl-Hem exhibited both dose and laser irradiation time dependent bactericidal effect on planktonic and biofilms of S. epidermidis. These results clearly showed that highly potent Cl-Hem can treat resistant microbial infections, while allowing fluorescence detection at the same time. High biofilm reduction observed with combined aPDT/aPTT action of Cl-Hem together with its non-cytotoxic nature points out that Cl-Hem is a promising PS for antibacterial and antibiofilm treatments.

Antimicrobial Activity of Methylene Blue Associated with Photodynamic Therapy: In Vitro Study in Multi-Species Oral Biofilm.

The control of infectious diseases caused by biofilms is a continuing challenge for researchers due to the complexity of their microbial structures and therapeutic implications. Photodynamic therapy as an adjunctive anti-infective treatment has been described as a possible valid approach but has not been tested in polymicrobial biofilm models. This study evaluated the effect of photodynamic therapy in vitro with methylene blue (MB) 0.01% and red LEDs (λ = 660 nm, power density ≈ 330 mW/cm2, 2 mm distance from culture) on the metabolic activity and composition of a multispecies subgingival biofilm. Test Groups LED and MB + LED showed a more significant reduction in metabolic activity than the non-LED application group (~50 and 55%, respectively). Groups LED and MB equally affected (more than 80%) the total bacterial count in biofilms. No differences were noted in the bacterial biofilm composition between the groups. In vitro LED alone or the MB + LED combination reduced the metabolic activity of bacteria in polymicrobial biofilms and the total subgingival biofilm count.

Antibacterial activity of corydalis saxicola bunting total alkaloids against Porphyromonas gingivalis in vitro.

Aim: To investigate the antibacterial effects of Corydalis Saxicola bunting total alkaloid (CSBTA) on Porphyromonas gingivalis. Methods: SEM, chemical staining, RT-qPCR and ELISA were used to detect effects of CSBTA on P. gingivalis. Results: CSBTA treatment caused shrinkage and rupture of P. gingivalis morphology, decreased biofilm density and live bacteria in biofilm, as well as reduced mRNA expression of virulence genes hagA, hagB, kgp, rgpA and rgpB of P. gingivalis. Furthermore, NOK cells induced by CSBTA-treated P. gingivalis exhibited lower IL-6 and TNF-α expression levels. Conclusion: CSBTA is able to kill free P. gingivalis, disrupt the biofilm and weaken the pathogenicity of P. gingivalis. It has the potential to be developed as a drug against P. gingivalis infection.

Effects of erythromycin on biofilm formation and resistance mutation of Escherichia coli on pristine and UV-aged polystyrene microplastics

Microplastics (MPs) and antibiotics co-occur widely in the environment and pose combined risk to microbial communities. The present study investigated the effects of erythromycin on biofilm formation and resistance mutation of a model bacterium, E. coli, on the surface of pristine and UV-aged polystyrene (PS) MPs sized 1–2 mm. The properties of UV-aged PS were significantly altered compared to pristine PS, with notable increases in specific surface area, carbonyl index, hydrophilicity, and hydroxyl radical content. Importantly, the adsorption capacity of UV-aged PS towards erythromycin was approximately 8-fold higher than that of pristine PS. Biofilms colonizing on UV-aged PS had a greater cell count (5.6 × 108 CFU mg−1) and a higher frequency of resistance mutation (1.0 × 10−7) than those on pristine PS (1.4 × 108 CFU mg−1 and 1.4 × 10−8, respectively). Moreover, erythromycin at 0.1 and 1.0 mg L−1 significantly (p < 0.05) promoted the formation and resistance mutation of biofilm on both pristine and UV-aged PS. DNA sequencing results confirmed that the biofilm resistance was attributed to point mutations in rpoB segment of the bacterial genome. qPCR results demonstrated that both UV aging and erythromycin repressed the expression levels of a global regulator rpoS in biofilm bacteria, as well as two DNA mismatch repair genes mutS and uvrD, which was likely to contribute to increased resistance mutation frequency.

A laboratory perspective on Mycobacterium abscessus biofilm culture, characterization and drug activity testing.

Mycobacterium abscessus is an emerging opportunistic pathogen causing severe pulmonary infections in patients with underlying lung disease and cystic fibrosis in particular. The rising prevalence of M. abscessus infections poses an alarming threat, as the success rates of available treatment options are limited. Central to this challenge is the absence of preclinical in vitro models that accurately mimic in vivo conditions and that can reliably predict treatment outcomes in patients. M. abscessus is notorious for its association with biofilm formation within the lung. Bacteria in biofilms are more recalcitrant to antibiotic treatment compared to planktonic bacteria, which likely contributes to the lack of correlation between preclinical drug activity testing (typically performed on planktonic bacteria) and treatment outcome. In recent years, there has been a growing interest in M. abscessus biofilm research. However, the absence of standardized methods for biofilm culture, biofilm characterization and drug activity testing has led to a wide spectrum of, sometimes inconsistent, findings across various studies. Factors such as strain selection, culture medium, and incubation time hugely impact biofilm development, phenotypical characteristics and antibiotic susceptibility. Additionally, a broad range of techniques are used to study M. abscessus biofilms, including quantification of colony-forming units, crystal violet staining and fluorescence microscopy. Yet, limitations of these techniques and the selected readouts for analysis affect study outcomes. Currently, research on the activity of conventional antibiotics, such as clarithromycin and amikacin, against M. abscessus biofilms yield ambiguous results, underscoring the substantial impact of experimental conditions on drug activity assessment. Beyond traditional drug activity testing, the exploration of novel anti-biofilm compounds and the improvement of in vitro biofilm models are ongoing. In this review, we outline the laboratory models, experimental variables and techniques that are used to study M. abscessus biofilms. We elaborate on the current insights of M. abscessus biofilm characteristics and describe the present understanding of the activity of traditional antibiotics, as well as potential novel compounds, against M. abscessus biofilms. Ultimately, this work contributes to the advancement of fundamental knowledge and practical applications of accurate preclinical M. abscessus models, thereby facilitating progress towards improved therapies for M. abscessus infections.

Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Tetracyclic homoisoflavanoid (+)-brazilin: a natural product inhibits c-di-AMP-producing enzyme and Streptococcus mutans biofilms.

The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.

A Diagnostic-Driven Prospective Clinical Study Evaluating the Combination of an Antibiofilm Agent and Negative Pressure Wound Therapy.

Each year, millions of Americans develop truncal pressure ulcers (PUs) which can persist for months, years, or until the end of life. Despite the negative impact on quality of life and escalating costs associated with PUs, there is sparse evidence supporting validated and efficacious treatment options. As a result, treatment is based on opinion and extrapolation from other wound etiologies. The ideal reconstructive plan maximizes the patient's nutritional status, incorporates the basic tenets of wound bed preparation (debridement, offloading, proper moisture balance, reduction of bacterial burden), and employs diagnostics to guide therapeutic intervention. The use of combination therapies can potentially overcome several of the barriers to wound healing. Negative pressure wound therapy (NPWT), a commonly used modality in the management of PUs, facilitates healing by stimulating the formation of granulation tissue and promoting wound contraction; however, NPWT alone is not always effective. Clinical studies examining microbial bioburden in PUs determined that most ulcers contain bacteria at levels that impede wound healing (>104 CFU/g). Thus, we hypothesized that adding an anti-microbial agent to decrease both planktonic and biofilm bacteria in the wound would increase the efficacy of NPWT. In this prospective study, twenty patients with recalcitrant PUs that previously failed NPWT were treated with a biofilm-disrupting agent (Blast-X, Next Science, Jacksonville, FL, USA) in combination with NPWT. Fluorescence imaging was used to follow bacterial burden and guide therapy. In total, 45% of the PUs reduced in size over the course of the four-week study, with a resolution of bacterial fluorescence in the NPWT dressing and wound bed seen in an average of three weeks. The combination of an antibiofilm agent and NPWT reduced bacterial levels and improved wound healing in recalcitrant PUs.

How xylitol, gluten, and lactose change human gut microbiota Escherichia coli and Lactobacillus rhamnosus GG biofilm

The human gut microbiota is composed of many viruses, bacteria, and fungi. Escherichia coli representatives are facultative anaerobic bacteria in the colon that play a crucial role in the metabolism of lactose, vitamin synthesis, and immune system modulation. E. coli forms a biofilm on the epithelial cell surface of the intestine that can be modified by diet compounds, i.e., gluten, xylitol, lactose, and probiotics. In the present study, the impact of probiotic-derived Lactobacillus rhamnosus GG strain on non-pathogenic E. coli biofilm was examined. The mono- and multispecies biofilm was also treated with gluten, xylitol, and lactose. We used 96-well plates to obtain biofilm growth. Biofilm was stained using crystal violet. To evaluate the type of interaction in mono- and multispecies biofilm, a new formula was introduced: biofilm interaction ratio index (BIRI). To describe the impact of nutrients on biofilm formation, the biofilm formation impact ratio (BFIR) was calculated. The biofilms formed by both examined species are stronger than in monocultures. All the BIRI values were above 3.0. It was found that the monospecies biofilm of L. rhamnosus is strongly inhibited by gluten (84.5%) and the monospecies biofilm of E. coli by xylitol (85.5%). The mixed biofilm is inhibited by lactose (78.8%) and gluten (90.6%). The relations between bacteria in the mixed biofilm led to changes in biofilm formation by E. coli and L. rhamnosus GG. Probiotics might be helpful in rebuilding the gut microbiota after broad spectrum antibiotic therapy, but only if gluten and lactose are excluded from diet.

Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modelling and whole genome sequencing

ObjectiveHypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies. MethodsTwo CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed. ResultsBoth isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. ConclusionThe combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination.

Isolation, Identification, and Biological Characterization of Phage vB_KpnM_KpVB3 Targeting Carbapenem-Resistant Klebsiella pneumoniae ST11

This study aimed to isolate a phage capable of lysing carbapenem-resistant Klebsiella pneumoniae (CRKP) and to analyse its biological characteristics and whole-genome sequence. The phage was isolated and purified from the sewage. Transmission electron microscopy (TEM) was employed to observe the bacteriophage's morphology. Phenotypic characterization of the bacteriophages was determined. The genomic information was analysed. Evolutionary relationships were established through comparative genomics, proteomics, and phylogenetic analysis. The isolation of a virulent phage, named Klebsiella phage vB_KpnM_KpVB3, was notable for forming 6-7 mm transparent circular zones, each surrounded by a distinct halo. The phage had a head diameter of ca. 30 nm and a tail length of ca. 20 nm, being identified as a member of the Myoviridae family and the Caudovirales order. The optimal multiplicity of infection (MOI) was 0.00001, with an incubation period of 20 minutes and a lysis period of 60 minutes, and the number of released phages after lysis was 133±35 PFU/cell. The phage was relatively stable at temperatures ranging from 10°C to 40°C and at pH values ranging from 3 to 11. Its lytic efficiency against CRKP was 30.30%. It has been shown to be able to destroy the biofilm of host bacteria. The bacteriophage genome consists of double-stranded DNA (dsDNA) with a total length of 48,394 base pairs, a GC content of 48.99%, and 78 open reading frames (ORFs). The study resulted in the isolation vB_KpnM_KpVB3, a phage demonstrating potential therapeutic efficacy against infections caused by CRKP.