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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 biofilm disruption by Ficin.S. aureus (A) and S. epidermidis (B) were grown in BM broth for 72 h to form a rigid biofilm, the mature biofilms were gently washed by BM and a fresh BM broth was loaded. Ficin was added until final concentrations of 10, 100 or 1000 μg/ml and incubation was followed for 24 h. The residual biofilms were quantified by crystal-violet staining.
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f2: The biofilm disruption by Ficin.S. aureus (A) and S. epidermidis (B) were grown in BM broth for 72 h to form a rigid biofilm, the mature biofilms were gently washed by BM and a fresh BM broth was loaded. Ficin was added until final concentrations of 10, 100 or 1000 μg/ml and incubation was followed for 24 h. The residual biofilms were quantified by crystal-violet staining.

Mentions: Over decades, a number of proteolytic enzymes have been adopted in clinical practice as wound healing agents destroying the cell debris and necrotic tissues. Recently, several proteases were reported to exhibit anti-biofilm properties and to increase the susceptibility of biofilm-embedded bacterial cells to antibiotics23242526. We investigated whether Ficin is able to disrupt bacterial biofilms formed by S. aureus and S. epidermidis, the bacteria colonizing wounds and thus retarding wound healing30. To do so, the bacteria were grown in BM broth earlier developed3132 for 72 h on 24-well TC-treated plates that provided a representative and repeatable formation of the rigid biofilm strongly attached to the surfaces, in contrast to Müller-Hinton broth, Trypticase soy broth or LB-medium (Fig. 1). Next the plates were washed twice by fresh BM followed by incubation during 24 h in the fresh BM broth in the presence of Ficin at concentrations of 10, 100 and 1000 μg/ml, since the recommended concentrations of proteolytic enzymes used for wounds healing (like Trypsin and Chymotrypsin) are 1–2 mg/ml3334. Then, the culture liquid was discarded and the residual biofilms were quantified by crystal violet staining. The control wells were subjected to all procedures described above except the enzyme addition after the wash and medium replacement. Wells were stained with crystal violet and their absorbance was taken as 100%. Our data indicate that Ficin effectively destroyed the established 3-days old biofilms formed by both S.aureus and S.epidermidis which can be typically observed on wounds35 and cause nosocomial infections (Fig. 2). Even at 10 μg/ml of Ficin only ca. 55–65% of the initial biofilm mass remained as confirmed by crystal violet staining, and biofilms were almost completely eliminated at higher Ficin concentration (1000 μg/ml) (OD570 < 0.1). Remarkably, the other proteolytic enzymes such as trypsin or papain could decrease the staphylococcal biofilm for 20–30% only at 100 μg/ml and on 50–60% at 1000 μg/ml36 confirming higher efficiency of Ficin for the treatment of staphylococcal biofilms.


Targeting microbial biofilms using Ficin, a nonspecific plant protease
The biofilm disruption by Ficin.S. aureus (A) and S. epidermidis (B) were grown in BM broth for 72 h to form a rigid biofilm, the mature biofilms were gently washed by BM and a fresh BM broth was loaded. Ficin was added until final concentrations of 10, 100 or 1000 μg/ml and incubation was followed for 24 h. The residual biofilms were quantified by crystal-violet staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The biofilm disruption by Ficin.S. aureus (A) and S. epidermidis (B) were grown in BM broth for 72 h to form a rigid biofilm, the mature biofilms were gently washed by BM and a fresh BM broth was loaded. Ficin was added until final concentrations of 10, 100 or 1000 μg/ml and incubation was followed for 24 h. The residual biofilms were quantified by crystal-violet staining.
Mentions: Over decades, a number of proteolytic enzymes have been adopted in clinical practice as wound healing agents destroying the cell debris and necrotic tissues. Recently, several proteases were reported to exhibit anti-biofilm properties and to increase the susceptibility of biofilm-embedded bacterial cells to antibiotics23242526. We investigated whether Ficin is able to disrupt bacterial biofilms formed by S. aureus and S. epidermidis, the bacteria colonizing wounds and thus retarding wound healing30. To do so, the bacteria were grown in BM broth earlier developed3132 for 72 h on 24-well TC-treated plates that provided a representative and repeatable formation of the rigid biofilm strongly attached to the surfaces, in contrast to Müller-Hinton broth, Trypticase soy broth or LB-medium (Fig. 1). Next the plates were washed twice by fresh BM followed by incubation during 24 h in the fresh BM broth in the presence of Ficin at concentrations of 10, 100 and 1000 μg/ml, since the recommended concentrations of proteolytic enzymes used for wounds healing (like Trypsin and Chymotrypsin) are 1–2 mg/ml3334. Then, the culture liquid was discarded and the residual biofilms were quantified by crystal violet staining. The control wells were subjected to all procedures described above except the enzyme addition after the wash and medium replacement. Wells were stained with crystal violet and their absorbance was taken as 100%. Our data indicate that Ficin effectively destroyed the established 3-days old biofilms formed by both S.aureus and S.epidermidis which can be typically observed on wounds35 and cause nosocomial infections (Fig. 2). Even at 10 μg/ml of Ficin only ca. 55–65% of the initial biofilm mass remained as confirmed by crystal violet staining, and biofilms were almost completely eliminated at higher Ficin concentration (1000 μg/ml) (OD570 < 0.1). Remarkably, the other proteolytic enzymes such as trypsin or papain could decrease the staphylococcal biofilm for 20–30% only at 100 μg/ml and on 50–60% at 1000 μg/ml36 confirming higher efficiency of Ficin for the treatment of staphylococcal biofilms.

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