Targeting microbial biofilms using Ficin, a nonspecific plant protease

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.

Enzymatic degradation of biofilm by metalloprotease fromMicrobacteriumsp. SKS10

“…This plate was further incubated for 24 hours where a well containing only LB medium without protease was considered as control. Congo red solution (final concentration of 50 μ g/ml) was used to stain the preformed biofilm (Baidamshina et al, 2017).…”

“…Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotic (kanamycin) on S. aureus MTCC 11949 was evaluated using standard protocols (Baidamshina et al, 2017). MIC was evaluated as the lowest concentration of kanamycin that was sufficient to prevent visible bacterial growth (no turbidity) after 24 hours of incubation.…”

“…MBC was determined as the lowest concentration of kanamycin necessary to kill 99.9% of bacteria. This is based on subculturing of bacterial inoculum (5 μ l) from the MIC wells that showed no visible growth into 5 ml of fresh LB broth followed by incubation for 24 hours at 37 °C (Baidamshina et al, 2017) in a shaker incubator.…”

Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus - Pseudomonas aeruginosa dual-species biofilm

“…Fresh colony material was used to adjust an optical density to 0.5 McFarland (equivalent to 10 8 cells/mL) in 0.9 % NaCl solution that was used as a working suspension. For the biofilm assay the previously developed BM broth (glucose 5g, peptone 7g, MgSO 4 × 7H 2 O 2.0g and CaCl 2 × 2H 2 O 0.05g in 1.0 liter tap water) 57,71,84 where both S. aureus and P. aeruginosa formed rigid biofilms in 2 days was used. The mannitol salt agar (peptones 10g, meat extract 1g, NaCl 75g, D-mannitol 10g, agar-agar 12g in 1.0 liter tap water, Oxoid) and cetrimide agar (Sigma) were used to distinguish S. aureus and P. aeruginosa , respectively, in mixed cultures.…”

Advances and Future Prospects of Enzyme‐Based Biofilm Prevention Approaches in the Food Industry

“…Serine-, cysteine-, and metalloproteases have been widely used as competent biofilm-controlling agents (Hou et al, 2017;Kumar, 2008;Marcato-Romain, Pechaud, Paul, Girbal-Neuhauser, & Dossat-Letisse, 2012;Miao et al, 2011;Watters et al, 2016) in either natural or commercial forms. Recently, a novel nonspecific sulfhydryl plant protease, ficin, was successfully used to eliminate S. aureus and S. epidermidis biofilms (Baidamshina et al, 2017). Today, phase-derived proteins are widely used to control many food-associated biofilm-forming pathogens (Gutiérrez et al, 2016).…”