Natural products as antibiofilm agents have been increasingly reported for their promising application in different clinical and environmental settings. However, present crises are mainly dealing with pathogenic biofilms and its control strategy in clinical settings. Medicinal plants are traditionally used in the treatment of various ailments including fungal infections, and many phytocompounds find application in the treatment of pathogenic biofilms in oral and systemic infections. Since biofilms development is associated with enhanced protection of fungi against stress conditions, resistance to antifungal drug treatments as well as contributing in increase protection from the host defense system. In addition, biofilm associated chronic infections are difficult to treat because of the preseinse of multi-drug resistant bacteria. Further, limited efficacy against biofilms and their host toxicity has compelled the researchers for improved formulation and discovery of new natural antibiofilm agents. It is believed that natural product-based antibiofilm agents are safer than synthetic drugs. The present review mainly focuses on various antibiofilm agents from natural sources, i.e., phytochemicals, microbial origin, bio-surfactant, and natural peptide with special reference phytocompounds and its application against the most common fungal pathogen, Candida spp. This information could be exploited to improve the existing therapies using antibiofilm agents possessing different applications in health care and in the food industry, etc. in preventing pathogenic biofilms.

The fungal biofilm-based infections are resistant to the most of currently available antifungal medications and affect many people worldwide. The pathogenicity of invasive fungal infection is usually accompanied by increased adhesion and the development of complex biofilms. Despite the development of several antifungal drugs that target the fungal plasma membrane and cell wall, these pathogens have evolved novel defense mechanisms that make them resistant to standard treatment. Only a few antifungal drugs are effective against biofilm-based fungal infections, including echinocandins and liposomal formulations of amphotericin B. To combat fungal biofilms, new approaches using a variety of synthetic compounds are currently being investigated. In this chapter, a brief background on pathogenic fungal biofilms, existing antifungals and their resistance mechanisms were introduced. Synthetic products that are passing through the clinical trial phase and potential applications of combinational therapies targeting the virulence and tolerance mechanism of fungal biofilm are reviewed.

The present study aimed to evaluate Syzygium aromaticum (clove) plant extract, clove oil and eugenol for their antibacterial activity and their potential to eradicate bacterial biofilms alone and in combination with antibiotics.Anti-bacterial efficacy of S. aromaticum extract, clove oil and eugenol was evaluated as minimum inhibitory concentration (MIC) and subsequently sub-MICs was selected for inhibition of virulence factors against test bacterial strains.Biofilm cultivation and eradication was assayed using XTT reduction in 96-well microtiter plate.Checkerboard method was used to study the interaction between essential oils and antibiotics.Staphylococcus aureus MTCC3160, Staphylococcus epidermidis MTCC435, Staphylococcus sciuri (SC-01), Staphylococcus auricularis (SU-01) and Streptococcus mutans MTCC497 were found strong biofilm former among all the test bacterial strains.The potency of test agents was found in the order of eugenol > clove oil > S. aromaticum methanolic extract.Sub-MIC (0.5 × MIC) of clove oil and eugenol showed a significant reduction in cell surface hydrophobicity (p < 0.05) and hemolysin production in the test bacterial strains.Eugenol showed no increase in sessile MIC (SMIC) against S. auricularis (SU-01), S. epidermidis MTCC435 and S. mutans MTCC497 compared to planktonic MIC (PMIC).Antibiotics (vancomycin and azithromycin) exhibited upto 1000-folds increased in SMIC compared to PMIC against all the test bacterial strains.Synergy was observed between eugenol and antibiotics (vancomycin/azithromycin) against all the test bacterial strains in both planktonic and sessile mode.Highest synergy was exhibited between eugenol and azithromycin in planktonic mode (FICI value 0.141).Further, microscopy also confirmed the spectacular effect of combina-How to cite this paper:

In this study, the antibiofilm activity of essential oil compounds eugenol and thymol alone and in combination with fluconazole and vancomycin againstCandida albicansandStaphylococcus aureuspre-formed mixed biofilms was investigated. Strong biofilm forming strains ofC. albicans(CAJ-01 and CAJ-12) andS. aureusMTCC3160 were selected for this study. Sessile minimum inhibitory concentration (SMIC) of eugenol and thymol was increased only 2-4 folds against single and mixed biofilm cells. However, the SMIC of the antimicrobial drugs was increased several folds. The fractional inhibitory concentration index (FICI) study highlighted the synergy between phytocompounds and antimicrobial drugs (fluconazole and vancomycin) against single and mixed biofilms. Single and mixed biofilm cells were eradicated at lower doses of the phytocompounds and antimicrobial drugs in combination treatment. The highest synergy recorded between thymol and vancomycin with FICI value of 0.125 againstC. albicans(CAJ-01 and CAJ-12) andS. aureusMTCC3160 mixed biofilms. Scanning electron microscopy studies also displayed a prominent effect on the morphology of theCandidaand bacterial biofilm cells at 3.25 mu g/ml concentration of thymol in a combination approach. The synergy of phytocompounds with antimicrobial drugs highlights the promising potential of these agents to be used in combinational anti-infective therapy to combat single and mixed biofilms associated infections.

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BACKGROUND:Candida albicans is frequently associated with mixed infections of Streptococcus mutans in plaque biofilms. These pathogens under chemical interactions resulting in mixed biofilm development have turned it into a prevalent and costly oral disease, which is not successfully being treated by existing chemotherapeutics.HYPOTHESIS:Considering the need for newer drugs to overcome this challenge, the present study was aimed to investigate the efficacy of eugenol in inhibiting single and mixed biofilms of C. albicans and S. mutans.METHODS:The broth dilution assay was used to determine drug resistance in the test strains. Biofilm formation on polystyrene microtiter plate was studied by XTT reduction assay whereas biofilm development on glass coverslips was assessed using 0.1% crystal violet and visualised under light microscope. Single and mixed biofilms formed on glass coverslips in the presence and absence of eugenol was analysed by scanning electron microscopy.RESULTS:In our study, all the thirteen strains of C. albicans were resistant to fluconazole, itraconazole, ketoconazole, amphotericin B except C. albicans (CAJ-01) and C. albicans MTCC3017 which were sensitive to fluconazole. S. mutans MTCC497 was resistant to ampicillin, azithromycin, ceftriaxone and vancomycin. Among all the strains of C. albicans, CAJ-01, C. albicans ATCC90028 and C. albicans MTCC3017 formed strong biofilms and rest of the strains considered as moderate to weak biofilm formers. S. mutans MTCC497 was also formed strong biofilms. Eugenol showed concentration dependent anti-biofilm activity against single and mixed biofilms of C. albicans (CAJ-01) and S. mutans MTCC497. At sub-MIC of eugenol (100 μg/ml), the biofilm formation was 36.37% and 29.72% in CAJ-01 and S. mutans MTCC497, respectively, whereas 52.65% in mixed biofilms. The cell viability assay showed significant reduction (p < 0.05) in the log10 CFU/ml from 6.3 to 4.8 at 200 μg/ml of eugenol for CAJ-01, whereas, from 6.4 to 3.8 and 5.3 for S. mutans MTCC497 strains in single and mixed biofilms, respectively. Scanning electron microscopy showed the disruption of cell membrane and matrix structure in both single and mixed biofilms.CONCLUSIONS:Eugenol at sub-MICs effectively inhibited single and mixed biofilms formed by the drug resistant strains of two oral pathogens, C. albicans and S. mutans through multiple mode of action.

Role of biofilm in disease development and enhance tolerance to antifungal drugs among Candida species has necessitated search for new anti-fungal treatment strategy. Interference in pathogenic biofilm development by new antifungal compounds is considered as an attractive anti-infective strategy. Therefore, the objective of this study was to evaluate Thymus vulgaris essential oil and its major active compound, thymol for their potential to inhibit and eradicate biofilms alone and in combination with antifungal drugs against Candida spp. with especial reference to Candida tropicalis. Anti-candidal efficacy of T. vulgaris and thymol in terms of minimum inhibitory concentration (MIC) was first determined to select the sub-MICs against C. albicans and C. tropicalis. Biofilm formation in the presence and absence of test agents was determined in 96-well microtiter plate by XTT reduction assay and effect of essential oils at sub-MICs of the test agents on biofilm development on glass surface was analysed by light and scanning electron microscopy. Synergistic interaction between essential oils and antifungal drugs were studied by checkerboard method. Effect of sub-MIC of T. vulgaris (0.5×MIC) and thymol (0.5×MIC) on biofilm formation showed a significant reduction (P<0.05) in biofilms. Light microscopy and SEM studies revealed disaggregation and deformed shape of C. albicans biofilm cells and reduced hyphae formation in C. tropicalis biofilm cells at sub-MICs of thymol. Significant effect of T. vulgaris and thymol was also recorded on pre-formed biofilms of both C. albicans and C. tropicalis. T. vulgaris and thymol also showed synergy with fluconazole against both in planktonic and biofilm mode of growth of C. albicans and C. tropicalis. However, synergy with amphotericin B is clearly evident only in planktonic Candida cells. Thyme oil and thymol alone or in combination with antifungal drugs can act as promising antibiofilm agent against drug resistant strains of Candida species and needs further in vivo study to synergise its therapeutic efficacy.

The transition from planktonic to sessile mode of bacterial growth facilitates the survival in diverse environmental niches. Majority of bacterial infections involve biofilm formation. Pathogenic biofilms in hospital settings are linked to the survival and spread of pathogens among hospital patients and may result in increased hospitalization of patient. Since biofilms are difficult to eradicate through conventional antibiotic therapy, various strategies are applied to treat biofilm infection. Natural products including phytocompounds from medicinal plants have been reported as antibiofilm agent in the last decade. In this chapter a brief description on the role of biofilm in pathogenesis and their common control strategies have been illustrated followed by a recent update on the role of plant essential oils/phytocompounds as the promising antibiofilm agent. The probable mechanisms in controlling biofilm by phytocompounds are highlighted.