Targeting microbial biofilms using Ficin, a nonspecific plant protease

Diana R Baidamshina,Elena Y Trizna,Rawil F Fakhrullin,Valeriy G Artyukhov,Elvira V Rozhina,Airat R Kayumov,Mikhail I Bogachev,Farida S Akhatova,Marina G Holyavka

doi:10.1038/srep46068

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.

Highlights

Biofilms are formed by the surface-attached bacterial cells arranged into complex communal tertiary structures and embedded into an extracellular matrix[1,2]
We investigated whether Ficin is able to disrupt bacterial biofilms formed by S. aureus and S. epidermidis, the bacteria colonizing wounds and retarding wound healing[30]
The bacteria were grown in Basal medium (BM) broth earlier developed[31,32] 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)

Summary

Introduction

Biofilms are formed by the surface-attached bacterial cells arranged into complex communal tertiary structures and embedded into an extracellular matrix[1,2]. Destroying the biofilm matrix backbone, for example via enzymatic lysis, is an advantageous approach for biofilms eradication[6] Numerous bacterial enzymes, such as glycosidases, proteases, and DNases degrade various components of biofilms stimulating cells detachment and increasing cellular susceptibility to antimicrobials[11]. Dispersin B injection in combination with triclosan reduced the catheter colonization density by S. aureus in rabbits in vivo[14] Another glycoside hydrolase, alginate lyase, successfully enhanced the activity of aminoglycosides against P. aeruginosa biofilms both in vitro[15,16] and in vivo[17]. In this paper we show that Ficin (EC 3.4.22.3), a nonspecific sulfhydryl protease isolated from the latex of the Ficus tree, disrupts the staphylococcal biofilm backbone, significantly increasing the efficiency of conventional antibiotics

Methods

Results

Conclusion