Polymicrobial Biofilm Formation Research Articles

Metabolic interplay between Proteus mirabilis and Enterococcus faecalis facilitates polymicrobial biofilm formation and invasive disease.

Polymicrobial biofilms play an important role in the development and pathogenesis of CAUTI. Proteus mirabilis and Enterococcus faecalis are common CAUTI pathogens that persistently co-colonize the catheterized urinary tract and form biofilms with increased biomass and antibiotic resistance. In this study, we uncover the metabolic interplay that drives biofilm enhancement and examine the contribution to CAUTI severity. Through compositional and proteomic biofilm analyses, we determined that the increase in biofilm biomass stems from an increase in the protein fraction of the polymicrobial biofilm matrix. We further observed an enrichment in proteins associated with ornithine and arginine metabolism in polymicrobial biofilms compared to single-species biofilms. We show that L-ornithine secretion by E. faecalis promotes arginine biosynthesis in P. mirabilis, and that disruption of this metabolic interplay abrogates the biofilm enhancement we see in vitro and leads to significant decreases in infection severity and dissemination in a murine CAUTI model.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Synergistic Interactions between Pseudomonas aeruginosa and Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis as well as Candida tropicalis in the Formation of Polymicrobial Biofilms

The interactions between pathogens during infection and the impact of these interactions on drug effectiveness are poorly understood, making polymicrobial infections challenging to treat. During an infection, cross-interactions between bacteria and fungi can strengthen virulence mechanisms and affect how the disease develops. The purpose of this study is to determine how Pseudomonas aeruginosa interacts with Candida glabrata, Candida albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis in the development of polymicrobial biofilms. Pseudomonas aeruginosa, Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis isolates were used in this experimental investigation. After preparing a 0.5 Mc Farland suspension of each isolate, the gold standard for measuring biofilm was applied: the Tissue Plate Culture (TCP) method. After that, an ELISA reader with a wavelength of 595 nm was used to measure the optical density (OD) of the biofilm. SPSS 26.0 was then used for statistical analysis to compare the OD values between Pseudomonas aeruginosa that had not been exposed to Candida and those that had. Pseudomonas aeruginosa and Candida are found to interact synergistically if there is an increase in OD, and antagonistic interaction is discovered if there is a decrease in OD. In comparison to the group that was not exposed to Candida, Pseudomonas aeruginosa exposed to Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis showed an increase in the OD value of biofilm. Pseudomonas aeruginosa and Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis interact synergistically.

Exploring the Complexity of the Interaction between T. rubrum and S. aureus/S. epidermidis in the Formation of Polymicrobial Biofilms.

Dermatophytes associated with bacteria can lead to severe, difficult-to-treat infections and contribute to chronic infections. Trichophyton rubrum, Staphylococcus aureus, and Staphylococcus epidermidis can form biofilms influenced by nutrient availability. This study investigated biofilm formation by these species by utilizing diverse culture media and different time points. These biofilms were studied through scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), biomass, metabolic activity, and colony-forming units (CFUs). The results revealed that mixed biofilms exhibited high biomass and metabolic activity when cultivated in the brain heart infusion (BHI) medium. Both bacterial species formed mature biofilms with T. rubrum within 72 h, irrespective of media. The timing of bacterial inoculation was pivotal in influencing biomass and metabolic activity. T. rubrum's development within mixed biofilms depended on bacterial addition timing, while pre-adhesion influenced fungal growth. Bacterial communities prevailed initially, while fungi dominated later in the mixed biofilms. CLSM revealed 363 μm thick T. rubrum biofilms with septate, well-developed hyphae; S. aureus (177 μm) and S. epidermidis (178 μm) biofilms showed primarily cocci. Mixed biofilms matched T. rubrum's thickness when associated with S. epidermidis (369 μm), with few hyphae initially. Understanding T. rubrum and Staphylococcal interactions in biofilms advances antimicrobial resistance and disease progression knowledge.

Peri-Implantitis: The Biomaterial Biofilm-Associated Polymicrobial Infection could be the Main Cause of Dental Implant Failure

In the past few decades, dental implants have proven aesthetic outcomes to gain recognition as the best treatment options for replacing missing or failing teeth with the optimal partnership of the patient and the Prosthodontist/Implantologist/ Restorative Dentistry Specialist. Polymicrobial biofilm formation on titanium surfaces becomes the main risk factor for inflammatory processes on tissues surrounding implant devices coupled with bone loss, which leads to implant failure. In an era of evidence-based dental medicine, considering the past and present medical histories of the patient to rule out possible elevated risk implant failure becomes a cause for concern. Periodontitis and peri-implantitis are polymicrobial infectious diseases on one hand. On the other hand, both have hyper-immune responsiveness from the host. There is no consistency in the finding of peri-implantitis-associated microbiome due to methodological variations in metagenomic studies conducted to this date. Moreover, there is controversy over the difference in microbiome profiles among periimplantitis and periodontitis. Putative periodontal pathogens seem to flourish in peri-implants-affected sites, suggesting the increasing virulence of those oral pathogens in dysbiosis and immunosuppressive conditions. Peri-implantitis biomaterial biofilm-associated polymicrobial infection called peri-implantitis could be among the root causes of dental implant failure with remaining unresolved issues and intrigue intricacies. Against this backdrop, this opinion aims to provide novel insights into the potential of dental implantology to unravel the mysteries in microbiology of peri-implantitis with a view to resolving the problem, though the work is still in its infancy. A lot more rigorous research is warranted in this regard, not only to define the implant associated microbiome but also to find a trustworthy solution to biological implant failure at the dawn of antibiotic-coated biomaterial production.

Peri-Implantitis: The Biomaterial Biofilm-Associated Polymicrobial Infection could be the Main Cause of Dental Implant Failure

In the past few decades, dental implants have proven aesthetic outcomes to gain recognition as the best treatment options for replacing missing or failing teeth with the optimal partnership of the patient and the Prosthodontist/Implantologist/ Restorative Dentistry Specialist. Polymicrobial biofilm formation on titanium surfaces becomes the main risk factor for inflammatory processes on tissues surrounding implant devices coupled with bone loss, which leads to implant failure. In an era of evidence-based dental medicine, considering the past and present medical histories of the patient to rule out possible elevated risk implant failure becomes a cause for concern. Periodontitis and peri-implantitis are polymicrobial infectious diseases on one hand. On the other hand, both have hyper-immune responsiveness from the host. There is no consistency in the finding of peri-implantitis-associated microbiome due to methodological variations in metagenomic studies conducted to this date. Moreover, there is controversy over the difference in microbiome profiles among periimplantitis and periodontitis. Putative periodontal pathogens seem to flourish in peri-implants-affected sites, suggesting the increasing virulence of those oral pathogens in dysbiosis and immunosuppressive conditions. Peri-implantitis biomaterial biofilm-associated polymicrobial infection called peri-implantitis could be among the root causes of dental implant failure with remaining unresolved issues and intrigue intricacies. Against this backdrop, this opinion aims to provide novel insights into the potential of dental implantology to unravel the mysteries in microbiology of peri-implantitis with a view to resolving the problem, though the work is still in its infancy. A lot more rigorous research is warranted in this regard, not only to define the implant associated microbiome but also to find a trustworthy solution to biological implant failure at the dawn of antibiotic-coated biomaterial production.

The respiratory enzyme complex Rnf is vital for metabolic adaptation and virulence in Fusobacterium nucleatum.

This paper illuminates the significant question of how the oral commensal Fusobacterium nucleatum adapts to the metabolically changing environments of several extra-oral sites such as placenta and colon to promote various diseases as an opportunistic pathogen. We demonstrate here that the highly conserved Rhodobacter nitrogen-fixation complex, commonly known as Rnf complex, is key to fusobacterial metabolic adaptation and virulence. Genetic disruption of this Rnf complex causes global defects in polymicrobial interaction, biofilm formation, cell growth and morphology, hydrogen sulfide production, and ATP synthesis. Targeted metabolomic profiling demonstrates that the loss of this respiratory enzyme significantly diminishes catabolism of numerous amino acids, which negatively impacts fusobacterial virulence as tested in a preterm birth model in mice.

Implant Surface Technologies to Prevent Surgical Site Infections in Spine Surgery.

Spine surgeries are occurring more frequently worldwide. Spinal implant infections are one of the most common complications of spine surgery, with a rate of 0.7% to 11.9%. These implant-related infections are a consequence of surface polymicrobial biofilm formation. New technologies to combat implant-related infections are being developed as their burden increases; however, none have reached the market stage in spine surgery. Conferring antimicrobial properties to biomaterials relies on either surface coating (physical, chemical, or combined) or surface modification (physical, chemical, or combined). Such treatment can also result in toxicity and the progression of antimicrobial resistance. This narrative review will discuss "late-stage" antimicrobial technologies (mostly validated in vivo) that use these techniques and may be incorporated onto spine implants to decrease the burden of implant-related health care-acquired infections (HAIs). Successfully reducing this burden will greatly improve the quality of life in spine surgery. Familiarity with upcoming surface technologies will help spine surgeons understand the anti-infective strategies designed to address the rapidly worsening challenge of implant-related health care-acquired infections.

Treatment of Staphylococcus aureus and Candida albicans polymicrobial biofilms by phloroglucinol-gold nanoparticles

The co-isolation of Staphylococcus aureus and Candida albicans from host tissues and organs and their in vitro and in vivo interaction studies suggest a synergistic relationship in forming polymicrobial biofilms. In particular, during polymicrobial biofilm formation, S. aureus becomes coated in the extracellular matrix secreted by C. albicans, leading to enhanced resistance to antibiotics. Accordingly, understanding the interactions between S. aureus and C. albicans in polymicrobial biofilms is of utmost importance in establishing treatment strategies for polymicrobial infections. As an alternate technique, nanoparticles were used in this investigation to suppress polymicrobial biofilm. The current study aims to manufacture gold nanoparticles (AuNPs) using phloroglucinol (PG), a natural chemical, and test their inhibitory capabilities against S. aureus and C. albicans biofilms in standard and host-mimicking media (like saliva and sputum). PG-AuNPs have a spherical form with an average size of 46.71 ± 6.40 nm. The minimum inhibitory concentration (MIC) values differed when PG-AuNPs were evaluated in the standard and host-mimicking artificial media. The MIC of PG-AuNPs against S. aureus and C. albicans was 2048 μg/mL in both the standard and artificial sputum media. However, the MIC in saliva was only 128 μg/mL. The initial stage polymicrobial biofilm of S. aureus and C. albicans was dramatically decreased at the sub-MIC of PG-AuNPs in both standard and host-mimicking media. S. aureus and C. albicans mature polymicrobial biofilms were more effectively eliminated by MIC and sub-MIC of PG-AuNPs. This study indicates that PG-AuNPs have the ability to limit the formation of polymicrobial biofilms caused by bacterial and fungal diseases.

Postbiotics of the Lactiplantibacillus plantarum EIR/IF-1 Strain Show Antimicrobial Activity against Oral Microorganisms with pH Adaptation Capability

Postbiotics offer better properties than probiotics. This study investigated the antimicrobial activity of Lactiplantibacillus plantarum EIR/IF-1 postbiotics against pH-adaptive bacteria, namely Prevotella denticola, Fusobacterium nucleatum, and Streptococcus sanguinis. Cell-free culture media of L. plantarum EIR/IF-1 were used as postbiotics in either crude (acidic) or neutralized form to also understand non-pH-dependent antimicrobial potential. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and viable cell counts were determined for crude and neutralized postbiotics. Culture media adjusted to different pH values were also compared to adjusted media with postbiotics to understand the strength of organic acids in postbiotics. Antibiofilm activity of postbiotics was determined against polymicrobial biofilm formation. Finally, the toxicity of crude postbiotics was tested on human periodontal ligament fibroblast cells (hPDLFCs). MIC values of crude postbiotics were 12.5 mg/mL for all strains. F. nucleatum and P. denticola strains were sensitive to neutralized postbiotics after 48 h of incubation. Moreover, 12.5 and 25 mg/mL postbiotics inhibited biofilm formation and 2.5 mg/mL and lower concentrations of crude postbiotics showed no cytotoxicity in hPDLFCs. This study showed that postbiotics have antimicrobial activity against pH-adaptive oral bacteria and no cytotoxic effect on hPDLFCs depending on the dose. The non-acidic antimicrobial components of postbiotics could also enable their safe use in the oral cavity.

Living together: The role of Candida albicans in the formation of polymicrobial biofilms in the oral cavity.

The oral cavity of humans is colonized by diversity of microbial community, although dominated by bacteria, it is also constituted by a low number of fungi, often represented by Candida albicans. Although in the vast minority, this usually commensal fungus under certain conditions of the host (e.g., immunosuppression or antibiotic therapy), can transform into an invasive pathogen that adheres to mucous membranes and also to medical or dental devices, causing mucosal infections. This transformation is correlated with changes in cell morphology from yeast-like cells to hyphae and is supported by numerous virulence factors exposed by C. albicans cells at the site of infection, such as multifunctional adhesins, degradative enzymes, or toxin. All of them affect the surrounding host cells or proteins, leading to their destruction. However, at the site of infection, C. albicans can interact with different bacterial species and in its filamentous form may produce biofilms-the elaborated consortia of microorganisms, that present increased ability to host colonization and resistance to antimicrobial agents. In this review, we highlight the modification of the infectious potential of C. albicans in contact with different bacterial species, and also consider the mutual bacterial-fungal relationships, involving cooperation, competition, or antagonism, that lead to an increase in the propagation of oral infection. The mycofilm of C. albicans is an excellent hiding place for bacteria, especially those that prefer low oxygen availability, where microbial cells during mutual co-existence can avoid host recognition or elimination by antimicrobial action. However, these microbial relationships, identified mainly in in vitro studies, are modified depending on the complexity of host conditions and microbial dominance in vivo.

Staphylococcus aureus strains exhibit heterogenous tolerance to direct cold atmospheric plasma therapy

The global clinical and socioeconomic impact of chronic wounds is substantial. The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site. Infected wounds arise from an accumulation of microbial aggregates in the wound bed, leading to the formation of polymicrobial biofilms that can be largely resistant to antibiotic therapy. Therefore, it is essential for studies to identify novel therapeutics to alleviate biofilm infections. One innovative technique is the use of cold atmospheric plasma (CAP) which has been shown to possess promising antimicrobial and immunomodulatory properties. Here, different clinically relevant biofilm models will be treated with cold atmospheric plasma to assess its efficacy and killing effects. Biofilm viability was assessed using live dead qPCR, and morphological changes associated with CAP evaluated using scanning electron microscopy (SEM). Results indicated that CAP was effective against Candida albicans and Pseudomonas aeruginosa, both as mono-species biofilms and when grown in a triadic model system. CAP also significantly reduced viability in the nosocomial pathogen, Candida auris. Staphylococcus aureus Newman exhibited a level of tolerance to CAP therapy, both when grown alone or in the triadic model when grown alongside C. albicans and P. aeruginosa. However, this degree of tolerance exhibited by S. aureus was strain dependent. At a microscopic level, biofilm treatment led to subtle changes in morphology in the susceptible biofilms, with evidence of cellular deflation and shrinkage. Taken together, these results indicate a promising application of direct CAP therapy in combatting wound and skin-related biofilm infections, although biofilm composition may affect the treatment efficacy.

Inhibition of Polymicrobial Biofilms of Candida albicans-Staphylococcus aureus/Streptococcus mutans by Fucoidan-Gold Nanoparticles.

The polymicrobial proliferation and development of complex biofilm morphologies by bacterial and fungal pathogens in the host are some of the key factors contributing to the failure of antimicrobial treatments. The polymicrobial interaction of Candida albicans and some bacterial species has been extensively studied in both in vitro and in vivo model systems. Alternative strategies for disrupting polymicrobial interaction and biofilm formation are constantly needed. Among several alternative strategies, the use of nanoparticles synthesized using a natural product in the treatment of microbial infection has been considered a promising approach. The current study aimed to synthesize gold nanoparticles (AuNPs) using a natural product, fucoidan, and to test their efficacy against mono and duo combinations of fungal (Candida albicans) and bacterial (Staphylococcus aureus/Streptococcus mutans) biofilms. Several methods were used to characterize and study Fu-AuNPs, including UV-vis absorption spectroscopy, FTIR, FE-TEM, EDS, DLS, zeta potential, and XRD. The concentration-dependent inhibition of early-stage biofilms and the eradication of mature biofilms of single species of C. albicans, S. aureus, and S. mutans have been observed. Early biofilms of a dual-species combination of C. albicans and S. aureus/S. mutans were also suppressed at an increasing concentration of Fu-AuNPs. Furthermore, Fu-AuNPs significantly eradicated the established mature biofilm of mixed species. The treatment method proposed in this study, which involves the use of marine-bioinspired nanoparticles, is a promising and biocompatible agent for preventing the growth of polymicrobial biofilms of bacterial and fungal pathogens.

The quorum quenching enzyme Aii20J modifies in vitro periodontal biofilm formation.

Recent studies have revealed the presence of N-acyl-homoserine lactones (AHLs) quorum sensing (QS) signals in the oral environment. Yet, their role in oral biofilm development remains scarcely investigated. The use of quorum quenching (QQ) strategies targeting AHLs has been described as efficient for the control of pathogenic biofilms. Here, we evaluate the use of a highly active AHL-targeting QQ enzyme, Aii20J, to modulate oral biofilm formation in vitro. The effect of the QQ enzyme was studied in in vitro multispecies biofilms generated from oral samples taken from healthy donors and patients with periodontal disease. Subgingival samples were used as inocula, aiming to select members of the microbiota of the periodontal pocket niche in the in vitro biofilms. Biofilm formation abilities and microbial composition were studied upon treating the biofilms with the QQ enzyme Aii20J. The addition of the enzyme resulted in significant biofilm mass reductions in 30 - 60% of the subgingival-derived biofilms, although standard AHLs could not be found in the supernatants of the cultured biofilms. Changes in biofilm mass were not accompanied by significant alterations of bacterial relative abundance at the genus level. The investigation of 125 oral supragingival metagenomes and a synthetic subgingival metagenome revealed a surprisingly high abundance and broad distribution of homologous of the AHL synthase HdtS and several protein families of AHL receptors, as well as an enormous presence of QQ enzymes, pointing to the existence of an intricate signaling network in oral biofilms that has been so far unreported, and should be further investigated. Together, our findings support the use of Aii20J to modulate polymicrobial biofilm formation without changing the microbiome structure of the biofilm. Results in this study suggest that AHLs or AHL-like molecules affect oral biofilm formation, encouraging the application of QQ strategies for oral health improvement, and reinforcing the importance of personalized approaches to oral biofilm control.

Biofilm formation by the interaction of fungi (Candida tropicalis) with various bacteria

Acinetobacter baumannii) ABSTRACT: Background: Biofilms are composed of more than one species or often called polymicrobial biofilms, so research is needed on the formation of polymicrobial biofilms, especially between fungi and bacteria. This study aimed to analyze the total biomass and metabolic activity of biofilms formed from the interaction of fungi (Candida tropicalis) with bacteria (Staphylococcus aureus, MRSA, Pseudomonas aeruginosa, and Acinetobacter baumannii); Methods: Bacterial and fungal cultures were suspended in Tryptic Soy Broth (TSB) medium, then biofilm was cultivated in two 96-well microplates, then the microplates were incubated at 37 degrees Celsius for 48 hours. Detection of the biofilm will be carried out using the Crystal Violet assay and reduction of the tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), or MTT assay to obtain the Optical Density (OD) value by using ELISA reader; Results: The results of this study obtained significant data from both parameters, the highest average value was found in the C. tropicalis treatment group (2,412 ± 0.825 on the biomass test results; 1,525 ± 0.473 on the metabolic activity test results), while the lowest was seen in the S. aureus treatment group (0.512 ± 0.224 in the biomass test results; 0.978 ± 0.349 in the metabolic activity test results); and Conclusions: In the total biomass parameter, it was obtained that the biofilm biomass could be formed strongly in each treatment, while the results of the metabolic activity showed that each treatment was able to carry out cell proliferation.

Formation and Analysis of Mono-species and Polymicrobial Oral Biofilms in Flow-Cell Models.

The oral microbiota, which is known to include at least 600 different bacterial species, is found on the teeth and mucosal surfaces as multi-species communities or biofilms. The oral surfaces are covered with a pellicle of proteins absorbed from saliva, and biofilm formation is initiated when primary colonizers, which express surface adhesins that bind to specific salivary components, attachto the oral tissues. Further development then proceeds through co-aggregation of additional species. Over time, the composition of oral biofilms, which varies between different sites throughout the oral cavity, is determined by a combination of environmental factors such as the properties of the underlying surface, nutrient availability and oxygen levels, and bacterial interactions within the community. A complex equilibrium between biofilm communities and the host is responsible for the maintenance of a healthy biofilm phenotype (eubiosis). In the face of sustained environmental perturbation, however, biofilm homeostasis can break down giving rise to dysbiosis, which is associated with the development of oral diseases such as caries and periodontitis.In vitro models have an important part to play in increasing our understanding of the complex processes involved in biofilm development in oral health and disease, and the requirements for experimental system, microbial complexity, and analysis techniques will necessarily vary depending on the question posed. In this chapter we describe some current and well-established methods used in our laboratory for studying oral bacteria in biofilm models which can be adapted to suit the needs of individual users.

Effect of extracellular levanase SacC from Bacillus subtillis on two–species Staphylococcus aureus – Pseudomonas aeruginosa biofilms

The formation of polymicrobial biofilms significantly increases the resistance of bacteria in them to a wide range of antibiotics thus making their eradication challenging in infectious medicine. In recent years, the emergence of a large number of antibiotic-resistant strains has contributed to a significant increase in severe cases of infectious diseases. Natural compounds such as alkaloids, terpenes, bacterial peptides and enzymes have great potential in combating polymicrobial diseases, associated with the biofilm formation. At the same time, the enzymatic treatment of biofilms has a number of advantages, since the enzymes are nontoxic, do not cause resistance and can increase the effectiveness of existing antimicrobials. Here we show that recombinant extracellular levanase SacC can promote the destruction of two-species biofilms S. aureus – P. aeruginosa and increase the effectiveness of antibiotics against bacteria in the biofilm up to 16 times.

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy

Bacterial vaginosis (BV) affects approximately 26% of women of childbearing age globally, presenting with 3–5 times increased risk of miscarriage and two-fold risk of pre-term birth. Antibiotics (metronidazole and clindamycin) are typically employed to treat BV; however the success rate is low due to the formation of recalcitrant polymicrobial biofilms. As a novel therapeutic, promising results have been obtained in vitro using Gardnerella endolysins, although to date their efficacy has only been demonstrated against simple biofilm models.In this study, a four-species biofilm was developed consisting of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and Mobiluncus curtisii. Biofilms were grown in NYC III broth and treated using antibiotics and an anti-Gardnerella endolysin (CCB7.1) for 24 h. Biofilm composition, viability and structure were assessed using colony counts, live/dead qPCR and scanning electron microscopy.All species colonised biofilms to varying degrees, with G. vaginalis being the most abundant. Biofilm composition remained largely unchanged when challenged with escalated concentrations of conventional antibiotics. A Gardnerella-targeted endolysin candidate (CCB7.1) showed efficacy against several Gardnerella species planktonically, and significantly reduced viable G. vaginalis within polymicrobial biofilms at 1 to 4X pMIC (p < 0.05 vs. vehicle control).Collectively, this study highlights the resilience of biofilm-embedded pathogens against the currently used antibiotics and provides a polymicrobial model that allows for more effective pre-clinical screening of BV therapies. The Gardnerella-specific endolysin CCB7.1 demonstrated significant activity against G. vaginalis within polymicrobial biofilms, altering the overall community dynamic and composition.

Inhibition of Candida albicans and Staphylococcus epidermidis mixed biofilm formation in a catheter disk model system treated with EtOH-EDTA solution.

Microbial colonization and the formation of biofilms on catheter surfaces pose a great risk for medical-related infections. We aimed (a) to evaluate polymicrobial biofilm formation of Candida albicans and Staphylococcus epidermidis and (b) to investigate the inhibition and effects of ethanol (EtOH) and EtOH-EDTA solutions on biofilms. Catheter disks were made and used as a substrate for biofilm formation. Varying concentrations of EtOH and EtOH-EDTA solutions were compared in deterring biofilm formation. The EtOH-EDTA solutions were further tested to remove mature and preformed biofilms. Compared to their monospecies counterparts, biofilm concentration significantly increases when C. albicans is co-cultured with S. epidermidis. Moreover, all treatments with EtOH-EDTA solution significantly lowered biofilm formation compared to EtOH alone (P≤0.05). Lastly, biofilm was dramatically reduced when treated with 20%, 30%, 40%, and 50% EtOH-EDTA solutions (P≤0.05). Our findings suggest that biofilms become more resilient to treatment when formed by multiple organisms. Nonetheless, treatment with EtOH-EDTA is effective against these polymicrobial biofilms.

Fabrication and characterization of a 3D polymicrobial microcosm biofilm model using melt electrowritten scaffolds

The majority of current biofilm models or substrates are two-dimensional (2D) and support biofilm growth in the horizontal plane only. Three-dimensional (3D) substrates may support both horizontal and vertical biofilm growth. This study compared biofilm growth quantity and quality between highly porous 3D micrometric fibrous scaffolds and 2D film substrates fabricated from medical grade polycaprolactone (mPCL). Melt electrowriting (MEW), a high-resolution additive manufacturing technology, was employed to design orderly aligned fine (~12 μm) fibre-based 3D scaffolds, while 2D films were fabricated by a casting method. The 3D scaffolds with a controlled pore size of 100 and 250 μm and thickness of ~0.8 mm and 2D films were incubated in pooled saliva collected from six volunteers for 1, 2, 4, 7 and 10 days at 37 °C to facilitate polymicrobial biofilm formation. Crystal violet assay demonstrated greater biofilm biomass in 3D MEW scaffolds than in 2D films. Biofilm thickness in 3D scaffolds was significantly higher compared to the biofilm thickness in 2D films. Both biovolume and substratum coverage of the biofilms was higher in the 3D scaffolds compared to 2D films. Polymeric bridges, pores, and channels characteristic of biofilms could be demonstrated by scanning electron microscopy. 16S rRNA sequencing demonstrated that the polymicrobial biofilms in the 3D scaffolds were able to retain 60–70 % of the original inoculum microbiome after 4 days. The MEW-fabricated 3D fibrous scaffold is a promising substrate for supporting multidirectional biofilm growth and modelling of a polymicrobial microcosm.