Polymicrobial Biofilms Research Articles

Urethral Catheter Biofilms Reveal Plasticity in Bacterial Composition and Metabolism and Withstand Host Immune Defenses in Hypoxic Environment.

Biofilms composed of multiple microorganisms colonize the surfaces of indwelling urethral catheters that are used serially by neurogenic bladder patients and cause chronic infections. Well-adapted pathogens in this niche are Escherichia coli, Proteus, and Enterococcus spp., species that cycle through adhesion and multilayered cell growth, trigger host immune responses, are starved off nutrients, and then disperse. Viable microbial foci retained in the urinary tract recolonize catheter surfaces. The molecular adaptations of bacteria in catheter biofilms (CBs) are not well-understood, promising new insights into this pathology based on host and microbial meta-omics analyses from clinical specimens. We examined catheters from nine neurogenic bladder patients longitudinally over up to 6 months. Taxonomic analyses from 16S rRNA gene sequencing and liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based proteomics revealed that 95% of all catheter and corresponding urinary pellet (UP) samples contained bacteria. CB biomasses were dominated by Enterobacteriaceae spp. and often accompanied by lactic acid and anaerobic bacteria. Systemic antibiotic drug treatments of patients resulted in either transient or lasting microbial community perturbations. Neutrophil effector proteins were abundant not only in UP but also CB samples, indicating their penetration of biofilm surfaces. In the context of one patient who advanced to a kidney infection, Proteus mirabilis proteomic data suggested a combination of factors associated with this disease complication: CB biomasses were high; the bacteria produced urease alkalinizing the pH and triggering urinary salt deposition on luminal catheter surfaces; P. mirabilis utilized energy-producing respiratory systems more than in CBs from other patients. The NADH:quinone oxidoreductase II (Nqr), a Na+ translocating enzyme not operating as a proton pump, and the nitrate reductase A (Nar) equipped the pathogen with electron transport chains promoting growth under hypoxic conditions. Both P. mirabilis and E. coli featured repertoires of transition metal ion acquisition systems in response to human host-mediated iron and zinc sequestration. We discovered a new drug target, the Nqr respiratory system, whose deactivation may compromise P. mirabilis growth in a basic pH milieu. Animal models would not allow such molecular-level insights into polymicrobial biofilm metabolism and interactions because the complexity cannot be replicated.

Inhibition of polymicrobial biofilm formation by saw palmetto oil, lauric acid and myristic acid.

SummaryBiofilms are communities of bacteria, fungi or yeasts that form on diverse biotic or abiotic surfaces, and play important roles in pathogenesis and drug resistance. A generic saw palmetto oil inhibited biofilm formation by Staphylococcus aureus, Escherichia coli O157:H7 and fungal Candida albicans without affecting their planktonic cell growth. Two main components of the oil, lauric acid and myristic acid, are responsible for this antibiofilm activity. Their antibiofilm activities were observed in dual‐species biofilms as well as three‐species biofilms of S. aureus, E. coli O157:H7 and C. albicans. Transcriptomic analysis showed that lauric acid and myristic acid repressed the expressions of haemolysin genes (hla and hld) in S. aureus, several biofilm‐related genes (csgAB, fimH and flhD) in E. coli and hypha cell wall gene HWP1 in C. albicans, which supported biofilm inhibition. Also, saw palmetto oil, lauric acid and myristic acid reduced virulence of three microbes in a nematode infection model and exhibited minimal cytotoxicity. Furthermore, combinatorial treatment of fatty acids and antibiotics showed synergistic antibacterial efficacy against S. aureus and E. coli O157:H7. These results demonstrate that saw palmetto oil and its main fatty acids might be useful for controlling bacterial infections as well as multispecies biofilms.

Candida albicans SET3 Plays a Role in Early Biofilm Formation, Interaction With Pseudomonas aeruginosa and Virulence in Caenorhabditis elegans.

The yeast Candida albicans exhibits multiple morphologies dependent on environmental cues. Candida albicans biofilms are frequently polymicrobial, enabling interspecies interaction through proximity and contact. The interaction between C. albicans and the bacterium, Pseudomonas aeruginosa, is antagonistic in vitro, with P. aeruginosa repressing the yeast-to-hyphal switch in C. albicans. Previous transcriptional analysis of C. albicans in polymicrobial biofilms with P. aeruginosa revealed upregulation of genes involved in regulation of morphology and biofilm formation, including SET3, a component of the Set3/Hos2 histone deacetylase complex (Set3C). This prompted the question regarding the involvement of SET3 in the interaction between C. albicans and P. aeruginosa, both in vitro and in vivo. We found that SET3 may influence early biofilm formation by C. albicans and the interaction between C. albicans and P. aeruginosa. In addition, although deletion of SET3 did not alter the morphology of C. albicans in the presence of P. aeruginosa, it did cause a reduction in virulence in a Caenorhabditis elegans infection model, even in the presence of P. aeruginosa.

Invasion of Diabetic and Atherosclerotic Vessels Patients with Periopathogen Spirochete Treponema denticola in Iraqi Population

Both Periodontitis & Atherosclerosis of Cardiovascular system are chronic inflammatory disorders can beperformed by invasion of their connective tissues with aerobic & anaerobic infectious bacteria & formationof polymicrobial biofilms. Microbial & metabolic plaques are considered to be the initiating factor for manyinfectious & metabolic diseases, invasion of an oral & systemic organs with various opportunistic pathogenshave been reported in many populations. Several studies have been published Conventional & Real timePCR with 16S r RNA gene to improve the existence of the DNA molecules of the most invasive periodonticspathogens in atheromatous plaques of diabetic & cardiovascular blood vessels disease patients.In the present study, among 100 atherosclerotic & diabetic patients suffered from various degrees of ischemicheart disease, the DNA molecules of Treponema denticola was detected in (45/100)45% of atheromatousplaque samples significantly in male 28 /45 (62%) more than 17/45(38%) in female patients (p = 0.07) withno significant importance in cardiovascular & diabetic patients more than 50 years old 23/45(51%) whichwas approximate to 22/45(49%) in younger than 50 years old. As well as, T. denticola DNA molecules weresignificantly displayed in atheromatous plaques of type (1, 2) diabetic patients 35 /45 (78%) more than innon-diabetic group 10/45(22%) (p= 0.07) & T. denticola DNA molecules did not displayed in the controllergroup.In conclusion: the present cross sectional study demonstrated the invasion of atheromatous plaques ofcardiovascular atherosclerotic vessels & diabetic patients with highly aggressive anaerobic periodontalspirochetes T. denticola, these results improve the existence of additional risk factor that interfere withthe pathogenesis of periodontitis & genesis of cardiovascular atherosclerosis & may be correlated with theatherosclerosis & diabetes mellitus diseases patients in Iraqi population.

5-hydroxymethyl-2-furaldehyde impairs Candida albicans - Staphylococcus epidermidis interaction in co-culture by suppressing crucial supportive virulence traits

Polymicrobial biofilms involving fungal-bacterial interactions are stated to modulate host immune response and exhibit enhanced antibiotic resistance. In this milieu, clinically important opportunistic pathogens Candida albicans and Staphylococcus epidermidis associate synergistically and instigate implant and blood stream infections. Impediment of virulence traits that support successive pathogenic lifestyle and inter-kingdom interactions without altering the microbial growth represents an attractive alternate strategy. To accomplish this objective, 5-hydroxymethyl-2-furaldehyde (5HM2F), a reported antibiofilm agent against C. albicans, was considered for this study. 5HM2F significantly repressed the biofilm formation of S. epidermidis and mixed-species at 300 μg/mL and 400 μg/mL, respectively without modulating the growth. Microscopic analyses and phenotypic assays explicated the competency of 5HM2F to impede biofilm formation, hyphal growth, initial attachment, intercellular adhesion, and fungal-bacterial interaction. Further, 5HM2F greatly reduced the secreted hydrolases production. Reduced content of biofilm matrix components upon 5HM2F treatment was believed to be the underlying reason for enhanced antibiotic and/antifungal susceptibility. Additionally, qPCR analysis correlated well with in vitro bioassays wherein, 5HM2F was identified to repress the expression of important genes associated with hyphal morphogenesis, adhesion, biofilm formation and virulence in both mono-species and mixed-species. Reduced virulence and colonization of mono-species and mixed-species in 5HM2F treated Caenorhabditis elegans substantiated the antibiofilm and antivirulence potential of 5HM2F. Overall, this study proposes 5HM2F as a potent therapeutic candidate against single and mixed-species biofilm infections of C. albicans and S. epidermidis.

Approaches to Understanding Mechanisms of Dentilisin Protease Complex Expression in Treponema denticola.

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5’ to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

Repurposing Kinase Inhibitor Bay 11-7085 to Combat Staphylococcus aureus and Candida albicans Biofilms.

Staphylococcus aureus and Candida spp. are commonly linked with topical biofilm-associated infections such as those found on chronic wounds. These biofilms are notoriously difficult to treat, highlighting the grave need to discover and study new broad-spectrum agents to combat associated infections. Here we report that the kinase inhibitor Bay 11-7085 exhibited bactericidal activity against multidrug-resistant S. aureus with a minimum inhibitory concentration (MIC) of 4 μg/ml. In addition, S. aureus strain MW2 did not acquire resistance to antibiotic pressure. Furthermore, Bay 11-7085 exhibited potency against Candida albicans and the emerging pathogen Candida auris with a MIC of 0.5–1 μg/ml. Bay 11-7085 partially inhibited and eradicated biofilm formation of various pathogens, such as VRSA (vancomycin-resistant S. aureus), as well as antifungal-resistant Candida spp. isolates. Notably, Bay 11-7085 partially inhibited initial cell attachment and formation of a VRSA-C. albicans polymicrobial biofilm in vitro. In contrast to C. albicans, inhibition of VRSA biofilm was linked to initial cell attachment independent of its bactericidal activity. Finally, Bay 11-7085 was effective in vivo at increasing the lifespan of C. elegans during an S. aureus and a C. albicans infection. Our work proposes kinase inhibitor Bay 11-7085 as a potential compound capable of combating biofilms associated with primary multidrug-resistant bacteria and yeast pathogens associated with wound infections.

In vitro polymicrobial inter-kingdom three-species biofilm model: influence of hyphae on biofilm formation and bacterial physiology

Biofilms are an important medical burden, notably for patients with orthopaedic device-related infections. When polymicrobial, these infections are more lethal and recalcitrant. Inter-kingdom biofilm infections are poorly understood and challenging to treat. Here, an in vitro three-species model including Staphylococcus aureus, Escherichia coli and Candida albicans was developed, to represent part of the diversity observed in orthopaedic infections or other clinical contexts. The importance of fungal hyphae for biofilm formation and virulence factor expression was explored. Two protocols were set up, allowing, or not, for hyphal formation. Culturable cells and biomass were characterised in both models, and biofilms were imaged in bright-field, confocal and electron microscopes. The expression of genes related to virulence, adhesion, exopolysaccharide synthesis and stress response was analysed in early-stage and mature biofilms. It was found that biofilms enriched in hyphae had larger biomass and showed higher expression levels of genes related to bacterial virulence or exopolysaccharides synthesis.

Supplemental nutrients stimulate the amplification of carbapenemase-producing Klebsiella pneumoniae (CPKP) in a sink drain in vitro biofilm reactor model

Liquid wastes (LW) disposed in hospital handwashing sinks may affect colonization of sink P-traps by carbapenemase-producing Klebsiella pneumoniae (CPKP), causing CPKP dispersal into the patient care environment. This study aimed to determine the effect of LW on biofilm formation and CPKP colonization in a P-Trap model (PTM). PTMs containing polymicrobial biofilms grown in autoclaved municipal tap water (ATW) supplemented with 5% dextrose in water (D5W), nutritional shake (Shake), sugar-based soft drink (Soda), or ATW were inoculated with K. pneumoniae ST258 KPC+ (ST258) or K. pneumoniae CAV1016 (CAV1016) and sampled after 7, 14, and 21 d. Biofilm bio-volume, mean thickness, and heterotrophic plate counts were significantly reduced and roughness coefficient significantly increased by Soda compared with D5W, Shake, or ATW. CPKP were significantly reduced by Soda but significantly amplified by D5W (ST258; CAV1016, 7 d) and Shake (ST258) suggesting that reducing LW disposal in sinks may reduce CPKP dispersal into patient care environments.

Targeting Candida albicans in dual-species biofilms with antifungal treatment reduces Staphylococcus aureus and MRSA in vitro.

Polymicrobial biofilms consisting of fungi and bacteria are frequently formed on endotracheal tubes and may contribute to development of ventilator associated pneumonia (VAP) in critically ill patients. This study aimed to determine the role of early Candida albicans biofilms in supporting dual-species (dual-kingdom) biofilm formation with respiratory pathogens in vitro, and investigated the effect of targeted antifungal treatment on bacterial cells within the biofilms. Dual-species biofilm formation between C. albicans and three respiratory pathogens commonly associated with VAP (Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was studied using quantitative PCR. It was shown that early C. albicans biofilms enhanced the numbers of E. coli and S. aureus (including methicillin resistant S. aureus; MRSA) but not P. aeruginosa within dual-species biofilms. Transwell assays demonstrated that contact with C. albicans was required for the increased bacterial cell numbers observed. Total Internal Reflection Fluorescence microscopy showed that both wild type and hyphal-deficient C. albicans provided a scaffold for initial bacterial adhesion in dual species biofilms. qPCR results suggested that further maturation of the dual-species biofilm significantly increased bacterial cell numbers, except in the case of E.coli with hyphal-deficient C. albicans (Ca_gcn5Δ/Δ). A targeted preventative approach with liposomal amphotericin (AmBisome®) resulted in significantly decreased numbers of S. aureus in dual-species biofilms, as determined by propidium monoazide-modified qPCR. Similar results were observed when dual-species biofilms consisting of clinical isolates of C. albicans and MRSA were treated with liposomal amphotericin. However, reductions in E. coli numbers were not observed following liposomal amphotericin treatment. We conclude that early C. albicans biofilms have a key supporting role in dual-species biofilms by enhancing bacterial cell numbers during biofilm maturation. In the setting of increasing antibiotic resistance, an important and unexpected consequence of antifungal treatment of dual-species biofilms, is the additional benefit of decreased growth of multi-drug resistant bacteria such as MRSA, which could represent a novel future preventive strategy.

The Female Vaginal Microbiome in Health and Bacterial Vaginosis.

The vaginal microbiome is an intricate and dynamic microecosystem that constantly undergoes fluctuations during the female menstrual cycle and the woman’s entire life. A healthy vaginal microbiome is dominated by Lactobacillus which produce various antimicrobial compounds. Bacterial vaginosis (BV) is characterized by the loss or sharp decline in the total number of Lactobacillus and a corresponding marked increase in the concentration of anaerobic microbes. BV is a highly prevalent disorder of the vaginal microbiota among women of reproductive age globally. BV is confirmed to be associated with adverse gynecologic and obstetric outcomes, such as sexually transmitted infections, pelvic inflammatory disease, and preterm birth. Gardnerella vaginalis is the most common microorganism identified from BV. It is the predominant microbe in polymicrobial biofilms that could shelter G. vaginalis and other BV-associated microbes from adverse host environments. Many efforts have been made to increase our understanding of the vaginal microbiome in health and BV. Thus, improved novel and accurate diagnosis and therapeutic strategies for BV have been developed. This review covers the features of vaginal microbiome, BV, BV-associated diseases, and various strategies of diagnosis and treatment of BV, with an emphasis on recent research progresses.

Dermal fibroblast cells interactions with single and triple bacterial-species biofilms.

Polymicrobial biofilm leads to wound healing delay. We set up an in vitro co-culture model of single- and triple-species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis with dermal fibroblast to assess the fibroblast response against to the different biofilms. Scratch and viability assays and biofilm cell quantifications were performed by WST-1, CLSM and plating method, respectively. Quorum sensing-related gene expression levels in P. aeruginosa and E. faecalis were analysed by reverse-transcriptase PCR. The immune responses of cells against S. aureus, P. aeruginosa and E. faecalis biofilms were measured by cytokine and matrix metalloproteinase analyzes. The influence of biofilm soluble factors on fibroblasts was also determined. After 24h, triple-species biofilm cells caused the removal of the fibroblasts from the surfaces indicating the negative synergistic effect of three species. After co-cultures, twenty-five cytokines were significantly increased in fibroblast cells compared to control. Compared to other strains, the most important cytokine, chemokine and growth factors increased was observed in P. aeruginosa co-cultures with fibroblast. While the expressions of fsrB and gelE genes were significantly upregulated in E. faecalis biofilm cells cultured with fibroblast cells, no significant difference was observed in P. aeruginosa. The wound healing and cell growth of fibroblasts were disrupted more aggressively in the presence of P. aeruginosa and triple-species biofilm cells. P. aeruginosa generally induced a stronger immune response in the fibroblasts than E. faecalis and S. aureus.

Multiple surface interaction mechanisms direct the anchoring, co-aggregation and formation of dual-species biofilm between Candida albicans and Helicobacter pylori

IntroductionPolymicrobial biofilms have a significant impact on pathogenesis of infectious microorganisms. Many human diseases are affected by colonization of multi-species communities affecting negatively the treatments and increase the risks for the health. In particular, in the epithelium of the stomach co-existence between C. albicans and H. pylori has been described, which has been associated to a synergistic effect on ulcer pathogenesis. ObjectiveThe objective of this work was to advance in the understanding of surface interaction between H. pylori and C. albicans for the formation of polymicrobial biofilms. MethodsStudies of microbial surfaces both bacterium, yeast and co-cultures of them were carried out by infrared spectroscopy, deconvolution analysis, transmission and scanning electron microscopies, and optic microscopy. Additional methods were used to contrast the results as dynamic light scattering, contact angle, agarose gel electrophoresis and gene amplification. ResultsSeveral surface interaction mechanisms promote the anchoring of H. pylori on C. albicans, cell co-aggregation, and polymicrobial biofilm formation, main identified interactions were: (i) hydrophobic interactions between non-polar peptide chains and lipid structures, characterized by θw among 84.9 ± 1.6 (γ = 22.78 mJ/m2 with 95.3 of dispersive contribution) and 76.6 ± 3.8 (γ = 17.34 mJ/m2, 40.2 of dispersive contribution) for C. albicans and H. pylori, respectively, (ii) hydrogen bonds between surface components of yeast and bacterium (e.g., SH⋅⋅⋅NH2 or SH⋅⋅⋅OCO) and (iii) thiol-mediated surface interactions identified by displacements to lower wavenumbers (Δv = 5 cm−1). Evidence of internalization and electrostatic interactions were not evidenced. All observations were congruent with the biofilm formation, including the identification of small-size biostructures (i.e., 122–459 nm) associated with extracellular proteins, extracellular DNA, or outer membrane vesicles were observed characteristic of biofilm formation. ConclusionIt is concluded that biofilm is formed by co-aggregation after anchoring of H. pylori on C. albicans. Several surface interactions were associated with the prevalence of H. pylori, the possibility to find C. albicans in the stomach epithelium infected by H. pylori, but also, strength interactions could be interfering in experimental observations associated with bacterial-DNA detection in culture mixtures.

Efficacy of Topical Vancomycin- and Gentamicin-Loaded Calcium Sulfate Beads or Systemic Antibiotics in Eradicating Polymicrobial Biofilms Isolated from Diabetic Foot Infections within an In Vitro Wound Model.

ABSTRACTDiabetic foot ulcers are notoriously difficult to heal, with ulcers often becoming chronic, in many cases leading to amputation despite weeks or months of antibiotic therapy in addition to debridement and offloading. Alternative wound biofilm management options, such as topical rather than systemic delivery of antimicrobials, have been investigated by clinicians in order to improve treatment outcomes. Here, we collected blood and tissue from six subjects with diabetic foot infections, measured the concentrations of antibiotics in the samples after treatment, and compared the microbiota within the tissue before treatment and after 7 days of antibiotic therapy. We used an in vitro model of polymicrobial biofilm infection inoculated with isolates from the tissue we collected to simulate different methods of antibiotic administration by simulated systemic therapy or topical release from calcium sulfate beads. We saw no difference in biofilm bioburden in the models after simulated systemic therapy (representative of antibiotics used in the clinic), but we did see reductions in bioburden of between 5 and 8 logs in five of the six biofilms that we tested with topical release of antibiotics via calcium sulfate beads. Yeast is insensitive to antibiotics and was a component of the sixth biofilm. These data support further studies of the topical release of antibiotics from calcium sulfate beads in diabetic foot infections to combat the aggregate issues of infectious organisms taking the biofilm mode of growth, compromised immune involvement, and poor systemic delivery of antibiotics via the bloodstream to the site of infection in patients with diabetes.

Modern understanding of bacterial vaginosis

Bacterial vaginosis is an infectious non-inflammatory syndrome characterized by the replacement of the usual microflora by the polymicrobial associations of anaerobes and Gardnerella vaginalis. The review is devoted to current issues of this disease, including pathogenesis, clinical manifestations and approaches to laboratory diagnostics. Discussion issues of this disease related to the possibility of sexual transmission are discussed. Particular attention is paid to the factors causing the development of bacterial vaginosis. The classical concepts of diagnostic criteria for diagnosis are presented. Bacterial vaginosis is not characterized by the presence of a specific microorganism, but is the result of the formation of a bacterial biofilm on the epithelium of the vagina or foreskin. The main component of this biofilm is G. vaginalis. Bacterial vaginosis is caused by polymicrobial gardnerella biofilm, which can only be transferred as a whole (for example, with the help of key cells) only during sexual intercourse. Along with this, the authors discuss modern aspects related to the problem of microbial biofilm formation. In this regard, the authors propose to use a new term biofilm vaginosis, which more accurately reflects the essence of this problem. The modern possibilities of treating bacterial vaginosis are presented. The use of combined local therapy of nonspecific inflammatory processes of the vulvovaginal zone is becoming especially relevant at the present stage. The use of a local combination drug (Gainomax) with the widest possible spectrum of action allows doctors to achieve rapid relief of symptoms and high efficiency of therapy.

Enzymatic biofilm destabilisation to support mechanical cleansing of inserted dental implant surfaces: an in-vitro pilot study

Peri-implantitis is caused by microbial contamination and biofilm formation on the implant surface. To achieve re-osseointegration, the microbes must be completely removed from the surface. Adjunctive to mechanical cleaning, chemical treatment with enzymes or other substances could optimise the treatment outcome. Therefore, we investigated the efficacy of different enzymes, a surfactant, and a chelator in destabilising dental polymicrobial biofilm. The biofilm destabilising effect of the glycosidases α-amylase, dextranase, DispersinB®, and lysozyme, as well as the proteinase subtilisin A, and the nuclease Benzonase®, the chelator EDTA, and the surfactant cocamidopropyl betaine were investigated on biofilms, inoculated with plaque on rough titanium discs. The test and the control solutions were incubated for 15 min at 36 °C on biofilms, and loosened biofilm mass was removed by shear stress with a shaker. Fluorescence-stained biofilms were microscopically analysed. Acceptable cell tolerability concentrations of test substances were determined by the MTT (tetrazolium dye) assay on the MG-63 cell line. A statistically significant biofilm destabilising effect of 10% was shown with lysozyme (2500 µg/ml).

Dual Function of Silver Nanoparticles as Matrix Extracell Removal and Antimicrobial Agent in Polymycrobial Biofilms

Candida albicans often form polymicrobial biofilms along with pathogenic microbes. Silver nanoparticles (AgNPs) were well known to have strong antimicrobial activity. However, their effect on polymicrobial biofilms and the mechanism has never been reported. This study aimed to synthesize AgNPs and study their effects on polymicrobial biofilm represented by C. albicans–E. coli biofilm. Polymicrobial biofilms, formed by clinical isolates of C. albicans and E. coli, were developed from the standardized suspensions of each strain by culturing flat-bottom 96-well microtiter plates for 48 h, then treated with AgNPs. Cell viability was assessed using the tetrazolium salt reduction assay; the extent of biofilm formation was measured by crystal violet staining. AgNPs reduced the polymicrobial biofilm in two ways: by degrading the extracellular matrix and killing both C. albicans and E. coli. The results showed AgNPs is a potential new approach for developing potent anti-biofilms.

Maintaining homeostatic control of periodontal bone tissue.

Alveolar bone is a unique osseous tissue due to the proximity of dental plaque biofilms. Periodontal health and homeostasis are mediated by a balanced host immune response to these polymicrobial biofilms. Dysbiotic shifts within dental plaque biofilms can drive a proinflammatory immune response state in the periodontal epithelial and gingival connective tissues, which leads to paracrine signaling to subjacent bone cells. Sustained chronic periodontal inflammation disrupts "coupled" osteoclast-osteoblast actions, which ultimately result in alveolar bone destruction. This chapter will provide an overview of alveolar bone physiology and will highlight why the oral microbiota is a critical regulator of alveolar bone remodeling. The ecology of dental plaque biofilms will be discussed in the context that periodontitis is a polymicrobial disruption of host homeostasis. The pathogenesis of periodontal bone loss will be explained from both a historical and current perspective, providing the opportunity to revisit the role of fibrosis in alveolar bone destruction. Periodontal immune cell interactions with bone cells will be reviewed based on our current understanding of osteoimmunological mechanisms influencing alveolar bone remodeling. Lastly, probiotic and prebiotic interventions in the oral microbiota will be evaluated as potential noninvasive therapies to support alveolar bone homeostasis and prevent periodontal bone loss.

Therapeutic implications of C. albicans-S. aureus mixed biofilm in a murine subcutaneous catheter model of polymicrobial infection

ABSTRACT Biofilm-associated polymicrobial infections tend to be challenging to treat. Candida albicans and Staphylococcus aureus are leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for in vivo-grown polymicrobial biofilms to validate our in vitro findings on C. albicans-mediated enhanced S. aureus tolerance to vancomycin in vivo. Comparative assessment of S. aureus recovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against S. aureus in mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. C. albicans induced the activation of the S. aureus biofilm formation network via down-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. In vitro follow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of C. albicans-mediated enhanced eDNA production in mixed-biofilms on S. aureus tolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies.

New Perspectives of Using Chitosan, Silver, and Chitosan–Silver Nanoparticles against Multidrug‐Resistant Bacteria

AbstractNanomaterials with antimicrobial activity are promising alternatives to overcome microbial resistance in medical devices. Catheters, probes, and wound dressings are among the medical devices mostly affected by microbial contamination and the formation of polymicrobial biofilms. Nanoparticles (NPs) derived from natural sources, such as chitosan nanoparticles (CsNPs), and metal‐based nanoparticles, including silver nanoparticles (AgNPs), are receiving increased interest in nanomedicine. CsNPs have been widely explored as a coating material and antimicrobial agent. AgNPs have a strong antimicrobial effect against bacteria and fungi. The nanocomposite chitosan–silver nanoparticles (Cs‐AgNPs) can be more effective against several microorganisms, including multidrug‐resistant bacteria, due to the synergistic effect between chitosan and silver. This review addresses the most used synthesis methods, including green routes, to produce CsNPs, AgNPs, and Cs–AgNPs. It also discusses physicochemical characteristics and antimicrobial properties of these NPs in medical, pharmaceutical, and biotechnological areas.