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Targeting microbial biofilms using Ficin, a nonspecific plant protease

View Article: PubMed Central - PubMed

ABSTRACT

Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24 hours treatment with Ficin at 10 μg/ml and six-fold at 1000 μg/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000 μg/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

No MeSH data available.


Related in: MedlinePlus

The Ficin treatment increases the efficacy of benzalkonium chloride against biofilm-embedded Staphylococci.Ficin (1000 μg/ml) and benzalkonium chloride (1–8 × MBC) were added to 48 hours-old biofilms of S. aureus and S. epidermidis. After 24 h incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended and their viability was analyzed by using drop plate assay (A,B). Alternatively, 48 hours-old biofilms of S. aureus and S. epidermidis were incubated 24 h in presence of Ficin (1000 μg/ml) and benzalkonium chloride (8 × MBC) in cell imaging coverglass slides and analyzed with confocal scanning microscopy (C–J). Significant differences between 10 log10 of the viable cell counts after treatment with benzalkonium chloride in either absence of presence of Ficin according to Pearson’s Chi-squared homogeneity test (p < 0.05) are indicated in the figure. The scale bars indicate 5 µm.
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f7: The Ficin treatment increases the efficacy of benzalkonium chloride against biofilm-embedded Staphylococci.Ficin (1000 μg/ml) and benzalkonium chloride (1–8 × MBC) were added to 48 hours-old biofilms of S. aureus and S. epidermidis. After 24 h incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended and their viability was analyzed by using drop plate assay (A,B). Alternatively, 48 hours-old biofilms of S. aureus and S. epidermidis were incubated 24 h in presence of Ficin (1000 μg/ml) and benzalkonium chloride (8 × MBC) in cell imaging coverglass slides and analyzed with confocal scanning microscopy (C–J). Significant differences between 10 log10 of the viable cell counts after treatment with benzalkonium chloride in either absence of presence of Ficin according to Pearson’s Chi-squared homogeneity test (p < 0.05) are indicated in the figure. The scale bars indicate 5 µm.

Mentions: The efficiency of other antimicrobials regularly used for outer treatment of wounds also increased in presence of Ficin. In particular, Ficin treatment led to the twofold decrease of the efficient concentration of Benzalkonium chloride, the biocide belonging to quaternary ammonium salts (Fig. 7, Fig. S3). Here, the significant differences between Ficin treated and untreated cells were observed at low concentrations of antimicrobial (1–2 × MBC) for both detached and biofilm-embedded cells. Similar effect could be observed for gentamycin (Fig. S4), although less pronounced, probably due to the low sensitivity of strains used to this antimicrobial.


Targeting microbial biofilms using Ficin, a nonspecific plant protease
The Ficin treatment increases the efficacy of benzalkonium chloride against biofilm-embedded Staphylococci.Ficin (1000 μg/ml) and benzalkonium chloride (1–8 × MBC) were added to 48 hours-old biofilms of S. aureus and S. epidermidis. After 24 h incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended and their viability was analyzed by using drop plate assay (A,B). Alternatively, 48 hours-old biofilms of S. aureus and S. epidermidis were incubated 24 h in presence of Ficin (1000 μg/ml) and benzalkonium chloride (8 × MBC) in cell imaging coverglass slides and analyzed with confocal scanning microscopy (C–J). Significant differences between 10 log10 of the viable cell counts after treatment with benzalkonium chloride in either absence of presence of Ficin according to Pearson’s Chi-squared homogeneity test (p < 0.05) are indicated in the figure. The scale bars indicate 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384253&req=5

f7: The Ficin treatment increases the efficacy of benzalkonium chloride against biofilm-embedded Staphylococci.Ficin (1000 μg/ml) and benzalkonium chloride (1–8 × MBC) were added to 48 hours-old biofilms of S. aureus and S. epidermidis. After 24 h incubation, the biofilms were washed twice with sterile 0.9% NaCl. The adherent cells were scratched, resuspended and their viability was analyzed by using drop plate assay (A,B). Alternatively, 48 hours-old biofilms of S. aureus and S. epidermidis were incubated 24 h in presence of Ficin (1000 μg/ml) and benzalkonium chloride (8 × MBC) in cell imaging coverglass slides and analyzed with confocal scanning microscopy (C–J). Significant differences between 10 log10 of the viable cell counts after treatment with benzalkonium chloride in either absence of presence of Ficin according to Pearson’s Chi-squared homogeneity test (p < 0.05) are indicated in the figure. The scale bars indicate 5 µm.
Mentions: The efficiency of other antimicrobials regularly used for outer treatment of wounds also increased in presence of Ficin. In particular, Ficin treatment led to the twofold decrease of the efficient concentration of Benzalkonium chloride, the biocide belonging to quaternary ammonium salts (Fig. 7, Fig. S3). Here, the significant differences between Ficin treated and untreated cells were observed at low concentrations of antimicrobial (1–2 × MBC) for both detached and biofilm-embedded cells. Similar effect could be observed for gentamycin (Fig. S4), although less pronounced, probably due to the low sensitivity of strains used to this antimicrobial.

View Article: PubMed Central - PubMed

ABSTRACT

Biofilms, the communities of surface-attached bacteria embedded into extracellular matrix, are ubiquitous microbial consortia securing the effective resistance of constituent cells to environmental impacts and host immune responses. Biofilm-embedded bacteria are generally inaccessible for antimicrobials, therefore the disruption of biofilm matrix is the potent approach to eradicate microbial biofilms. We demonstrate here the destruction of Staphylococcus aureus and Staphylococcus epidermidis biofilms with Ficin, a nonspecific plant protease. The biofilm thickness decreased two-fold after 24&thinsp;hours treatment with Ficin at 10&thinsp;&mu;g/ml and six-fold at 1000&thinsp;&mu;g/ml concentration. We confirmed the successful destruction of biofilm structures and the significant decrease of non-specific bacterial adhesion to the surfaces after Ficin treatment using confocal laser scanning and atomic force microscopy. Importantly, Ficin treatment enhanced the effects of antibiotics on biofilms-embedded cells via disruption of biofilm matrices. Pre-treatment with Ficin (1000&thinsp;&mu;g/ml) considerably reduced the concentrations of ciprofloxacin and bezalkonium chloride required to suppress the viable Staphylococci by 3 orders of magnitude. We also demonstrated that Ficin is not cytotoxic towards human breast adenocarcinoma cells (MCF7) and dog adipose derived stem cells. Overall, Ficin is a potent tool for staphylococcal biofilm treatment and fabrication of novel antimicrobial therapeutics for medical and veterinary applications.

No MeSH data available.


Related in: MedlinePlus