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Biofilms confer several advantages to the organisms associated with them, such as increased resistances to antibacterial and antifungal compounds compared to free living cells. Compared to monomicrobial biofilms involving a single microorganism, biofilms composed of microorganisms affiliated to bacterial and fungal kingdoms are predominant in nature. Despite the predominance of polymicrobial biofilms, and more so mixed polymicrobial biofilms, they are rarely studied. The objective of the current study is to evaluate the potential of ocular bacteria and a filamentous fungus to form monomicrobial and mixed polymicrobial biofilms on synthetic and natural substrates and to monitor their response to antibiotics. In this sense, we demonstrated that the ocular pathogens Staphylococcus aureus, S. epidermidis, and Fusarium solani form monomicrobial and mixed polymicrobial biofilms both on tissue culture polystyrene plates and on ex vivo human corneas from cadavers using confocal microscopy and scanning electron microscopy. Additionally, the mixed polymicrobial biofilms involving the above ocular bacteria and a filamentous fungus were less susceptible to different antibacterials and antifungals in relation to the corresponding control planktonic cells. Further, the MICs to the screened antibacterials and antifungals in polymicrobial biofilms involving a bacterium or a fungus was either increased, decreased, or unchanged compared to the corresponding individual bacterial or fungal biofilm. The results would be useful to the ophthalmologist to plan effective treatment regimens for the eye since these are common pathogens of the eye causing keratitis, endophthalmitis, conjunctivitis, etc.

Successful pathogens require metabolic flexibility to adapt to diverse host niches. The presence of co-infecting or commensal microorganisms at a given infection site can further influence the metabolic processes required for a pathogen to cause disease. The Gram-positive bacterium Staphylococcus aureus and the polymorphic fungus Candida albicans are microorganisms that asymptomatically colonize healthy individuals but can also cause superficial infections or severe invasive disease. Due to many shared host niches, S. aureus and C. albicans are frequently co-isolated from mixed fungal-bacterial infections. S. aureus and C. albicans co-infection alters microbial metabolism relative to infection with either organism alone. Metabolic changes during co-infection regulate virulence, such as enhancing toxin production in S. aureus or contributing to morphogenesis and cell wall remodeling in C. albicans . C. albicans and S. aureus also form polymicrobial biofilms, which have greater biomass and reduced susceptibility to antimicrobials relative to mono-microbial biofilms. The S. aureus and C. albicans metabolic programs induced during co-infection impact interactions with host immune cells, resulting in greater microbial survival and immune evasion. Conversely, innate immune cell sensing of S. aureus and C. albicans triggers metabolic changes in the host cells that result in an altered immune response to secondary infections. In this review article, we discuss the metabolic programs that govern host-pathogen interactions during S. aureus and C. albicans co-infection. Understanding C. albicans-S. aureus interactions may highlight more general principles of how polymicrobial interactions, particularly fungal-bacterial interactions, shape the outcome of infectious disease. We focus on how co-infection alters microbial metabolism to enhance virulence and how infection-induced changes to host cell metabolism can impact a secondary infection.

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria co-isolated from chronic infected wounds. Their interactions remain unclear but this coexistence is beneficial for both bacteria and may lead to resistance to antimicrobial treatments. Besides, developing an in vitro model where this coexistence is recreated remains challenging, making difficult their study. The aim of this work was to develop a reliable polymicrobial in vitro model of both species to further understand their interrelationships and the effects of different antimicrobials in coculture. In this work, bioluminescent and fluorescent bacteria were used to evaluate the activity of two antiseptics (chlorhexidine and thymol) against these bacteria planktonically grown, or when forming single and mixed biofilms. At the doses tested (0.4-1,000 mg/L), thymol showed selective antimicrobial action against S. aureus in planktonic and biofilm states, in contrast with chlorhexidine which exerted antimicrobial effects against both bacteria. Furthermore, the initial conditions for both bacteria in the co-culture determined the antimicrobial outcome, showing that P. aeruginosa impaired the proliferation and metabolism of S. aureus . Moreover, S. aureus showed an increased tolerance against antiseptic treatments when co-cultured, attributed to the formation of a thicker mixed biofilm compared to those obtained when monocultured, and also, by the reduction of S. aureus metabolic activity induced by diffusible molecules produced by P. aeruginosa. This work underlines the relevance of polymicrobial populations and their crosstalk and microenvironment in the search of disruptive and effective treatments for polymicrobial biofilms.

Hesperetin is the aglycone of citrus flavonoid hesperidin. Due to the limited information regarding hesperetin antimicrobial potential and emerging need for novel antimicrobials, we have studied its antimicrobial activity (microdilution assay), antibiofilm activity with different assays in two models (mono- and polymicrobial biofilm), and toxicity (MTT and brine shrimp lethality assays). Hesperetin inhibited growth of all Candida isolates (minimal inhibitory concentration, MIC, 0.165 mg/mL), while it’s inhibitory potential towards Staphylococcus aureus was lower (MIC 4 mg/mL). Hesperetin (0.165 mg/mL) reduced ability of Candida to form biofilms and moderately reduced exopolysaccharide levels in biofilm matrix. Effect on the eradication of 24 h old C. albicans biofilms was promising at 1.320 mg/mL. Inhibition of staphylococcal biofilm formation required higher concentrations of hesperetin (<50% inhibition with MIC 4 mg/mL). Establishment of polymicrobial C. albicans-S. aureus biofilm was significantly inhibited with the lowest examined hesperetin concentration (1 mg/mL) in crystal violet and CFU assays. Hesperetin toxicity was examined towards MRC-5 fibroblasts (IC50 0.340 mg/mL) and in brine shrimp lethality assay (LC50 > 1 mg/mL). Hesperetin is efficient in combating growth and biofilm formation of Candida species. However, its antibacterial application should be further examined due to the cytotoxic effects provoked in the antibacterial concentrations.

It is well known that the opportunistic pathogenic yeast, Candida albicans , can form polymicrobial biofilms with a variety of bacteria, both in vitro and in vivo , and that these polymicrobial biofilms can impact the course and management of disease. Although specific interactions are often described as either synergistic or antagonistic, this may be an oversimplification. Polymicrobial biofilms are complex two-way interacting communities, regulated by inter-domain (inter-kingdom) signaling and various molecular mechanisms. This review article will highlight advances over the last six years (2016-2021) regarding the unique biology of polymicrobial biofilms formed by C. albicans and bacteria, including regulation of their formation. In addition, some of the consequences of these interactions, such as the influence of co-existence on antimicrobial susceptibility and virulence, will be discussed. Since the aim of this knowledge is to inform possible alternative treatment options, recent studies on the discovery of novel anti-biofilm compounds will also be included. Throughout, an attempt will be made to identify ongoing challenges in this area.

Innovative non-antibiotic compounds such as graphene oxide (GO) and light-emitting diodes (LEDs) may represent a valid strategy for managing chronic wound infections related to resistant pathogens. This study aimed to evaluate 630 nm LED and 880 nm LED ability to enhance the GO antimicrobial activity against Staphylococcus aureus- and Pseudomonas aeruginosa-resistant strains in a dual-species biofilm in the Lubbock chronic wound biofilm (LCWB) model. The effect of a 630 nm LED, alone or plus 5-aminolevulinic acid (ALAD)-mediated photodynamic therapy (PDT) (ALAD-PDT), or an 880 nm LED on the GO (50 mg/l) action was evaluated by determining the CFU/mg reductions, live/dead analysis, scanning electron microscope observation, and reactive oxygen species assay. Among the LCWBs, the best effect was obtained with GO irradiated with ALAD-PDT, with percentages of CFU/mg reduction up to 78.96% ± 0.21 and 95.17% ± 2.56 for S. aureus and P. aeruginosa, respectively. The microscope images showed a reduction in the cell number and viability when treated with GO + ALAD-PDT. In addition, increased ROS production was detected. No differences were recorded when GO was irradiated with an 880 nm LED versus GO alone. The obtained results suggest that treatment with GO irradiated with ALAD-PDT represents a valid, sustainable strategy to counteract the polymicrobial colonization of chronic wounds.

Three-dimensional (3D) scaffold systems have proven instrumental in advancing our understanding of polymicrobial biofilm dynamics and probiotic interactions within the oral environment. Among oral probiotics, Streptococcus salivarius K12 (Ssk12) has shown considerable promise in modulating microbial homeostasis; however, its long-term therapeutic benefits are contingent upon successful and sustained colonization of the oral mucosa. Despite its clinical relevance, the molecular mechanisms underlying the adhesion, persistence, and integration of Ssk12 into the native oral microbiome/biofilm remain inadequately characterized. In this pilot study, we explored the temporal colonization dynamics of Ssk12 and its impact on the structure and functional profiles of salivary-derived biofilms cultivated on melt-electrowritten poly(ε-caprolactone) (MEW-mPCL) scaffolds, which emulate the native oral niche. Colonization was monitored via fluorescence in situ hybridization (smFISH), confocal microscopy, and RT-qPCR, while shifts in community composition and function were assessed using 16S rRNA sequencing and meta-transcriptomics. A single administration of Ssk12 exhibited transient colonization lasting up to 7 days, with detectable presence diminishing by day 10. This was accompanied by short-term increases in Lactobacillus and Bifidobacterium populations. Functional analyses revealed increased transcriptional signatures linked to oxidative stress resistance and metabolic adaptation. These findings suggest that even short-term probiotic colonization induces significant functional changes, underscoring the need for strategies to enhance probiotic persistence.

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 human microbiome is an assemblage of diverse bacteria that interact with one another to form communities. Bacteria in a given community are arranged in a 3D matrix with many degrees of freedom. Snapshots of the community display well-defined structures, but the steps required for their assembly are not understood. Here, we show that this construction is carried out with the help of gliding bacteria. Gliding is defined as the motion of cells over a solid or semisolid surface without the necessity of growth or the aid of pili or flagella. Genomic analysis suggests that gliding bacteria are present in human microbial communities. We focus on Capnocytophaga gingivalis, which is present in abundance in the human oral microbiome. Tracking of fluorescently labeled single cells and of gas bubbles carried by fluid flow shows that swarms of C. gingivalis are layered, with cells in the upper layers moving more rapidly than those in the lower layers. Thus, cells also glide on top of one another. Cells of nonmotile bacterial species attach to the surface of C. gingivalis and are propelled as cargo. The cargo cell moves along the length of a C. gingivalis cell, looping from one pole to the other. Multicolor fluorescent spectral imaging of cells of different live but nonmotile bacterial species reveals their long-range transport in a polymicrobial community. A swarm of C. gingivalis transports some nonmotile bacterial species more efficiently than others and helps to shape the spatial organization of a polymicrobial community.

Background Heat polymerized acrylic (HPA) resins are known to have high porosity that contributes to increased surface roughness and microcrack formation in stress areas. This facilitates the attachment and growth of polymicrobial biofilms contributing to increased antimicrobial resistance. Many research had been carried out on avocado seeds, but no research that studies avocado seeds effect on denture-plaque microorganism biofilm on HPA resin has been found. Methods This study used 144 samples (n=144), namely HPA resin discs covered with mono-species and polymicrobial biofilms. The discs were soaked for 8 hours in the 5%, 10%, 15%, 20% avocado seed extract, positive control (alkaline peroxide), and negative control (aquadest). Each disc was shaken with a vortex mixer for 1 minute, and 100 μL was added into 96-well microplates with three times repetition and incubated for 24 hours. A microtiter plate biofilm formation assay was then conducted The inhibition values were determined from the percentage inhibition value formula which required absorption values from a microplate reader (595 nm). The research data were analyzed using a univariant test, and a one-way ANOVA test, accompanied by Welch ANOVA on non-homogenous data. Results In this research, it was found that the MBIC 50 of avocado seed extract against the mono-species of C. albicans (5%), C. glabrata (5%), A. odontolyticus (15%), S. gordonii (15%), S. aureus (10%), while against polymicrobial was 20%. There was a significant effect of soaking HPA resin in avocado seed extract on the inhibition of mono-species and polymicrobial biofilms with a value of p<0.001 (p<0.05). Conclusion The MBIC 50 of avocado seed extract in polymicrobial biofilm group was higher than that in the mono-species biofilm groups. Although alkaline peroxide showed higher inhibition value than that of the MBIC 50 in polymicrobial biofilm group, 20% avocado seed extract was concluded effective as it inhibited >50% polymicrobial biofilm.