Carvacrol inhibits biofilm formation and production of extracellular polymeric substances of Pectobacterium carotovorum subsp. carotovorum

Highlights

• Carvacrol reduced swimming motility of Pectobacterium carotovorum.

• Carvacrol reduced biofilm formation of P. carotovorum at 0.66 mM.

• EPS synthesis was reduced by the presence of carvacrol.

• Polysaccharides are the main constituents of P. carotovorum biofilm matrix.

Abstract

Pectobacterium carotovorum causes soft rot in plant food due to its ability to synthesize exoenzymes, and secrete polymeric substances to form biofilm; in addition, this aggregated form of bacteria creates resistance during disinfection. However, the biofilm formation process and composition of P. carotovorum is still uncharacterized, which is important to design more efficient disinfection procedures. Among the actual disinfection trends, the use of plant compounds has deserved plenty of attention; being carvacrol, a monoterpene constituent of oregano essential oil that is known to reduce biofilm formation of many bacteria. Therefore, the objective of this study was to evaluate the effect of carvacrol against biofilm development of P. carotovorum, EPS composition, bacterial surface charge, adhesion potential and motility. Minimal planktonic inhibitory and bactericidal concentrations of carvacrol were 2.66 and 3.99 mM; whereas, concentrations of 1.33 and 3.99 mM were needed to inhibit and eradicate biofilm, respectively. Carvacrol at 0.66 mM was chosen as the concentration to evaluate its effect on EPS secretion, motility and surface physicochemical characteristics without affecting the viability of planktonic cells. It was shown that carvacrol at this concentration decreased bacterial surface charge (−2.15 mV), adhesion potential (−1.5 mJ/m²) and swimming motility of P. carotovorum (48.2 mm) compared with untreated bacteria. In addition, polysaccharides were the main components of the biofilm matrix of P. carotovorum, whose synthesis was inhibited by the presence of carvacrol. These results demonstrated that carvacrol could be effective against P. carotovorum biofilm formation and eradication, reducing adhesion, motility and synthesis of polysaccharides.

Introduction

Pectobacterium carotovorum subsp. carotovorum (before called Erwinia carotovora) is a plant pathogen that causes soft rot disease in cabbage, potato, onion, radish and other crops during cultivation, transportation and storage, resulting in high losses worldwide every year and considerable economic damage (Song et al., 2013). The ability of P. carotovorum to adhere and form biofilm is an important factor that contributes to its capacity to infect plant tissues, as well as to its resistance and persistence in different environments, especially in the field. However, this bacterium could be present also on surfaces during postharvest handling, being cause of cross-contamination and postharvest decay (Czajkowski, Pérombelon, van Veen, & van der Wolf, 2011).

A biofilm is a community of microorganisms embedded in a matrix of extracellular polymeric substances (EPS) of own production, mainly composed by polysaccharides, proteins, lipids and extracellular DNA, which can vary in composition among microorganisms and surrounding conditions (Flemming & Wingender, 2010). The biofilm formation of P. carotovorum is regulated by the quorum sensing (QS) system throughout the ExpI/R proteins. For most bacteria it generally happens in 5 main stages: 1) reversible adhesion, 2) irreversible adhesion, 3) microcolonies, 4) maturation and 5) dispersion (Phillips, Yang, Sampson, & Schultz, 2010). Bacterium uses flagella-mediated motility to overcome the repulsion forces between this and the target surface and once adhered they initiate their infection process, which is characterized for synthesis of plant cell wall degrading enzymes (pectate lyases, cellulases, proteases), as well as biofilm formation (Prigent-Combaret et al., 2012). At this point, the composition of P. carotovorum biofilms is still unknown; however, it has been observed that other plant pathogens have polysaccharides as main components of their biofilm matrixes, which are related to cell-cell, cell-surface adhesion and protection against disinfectants and other stresses (Flemming et al., 2016).

Several strategies for controlling bacterial adhesion to surfaces have been proposed, including the use of natural compounds. In this sense, essential oils and their main constituents have shown potential to inhibit bacterial cells in planktonic and sessile state (biofilms) (Ortega-Ramirez et al., 2017, Tapia-Rodriguez et al., 2017). Carvacrol, a hydrophobic terpene component of oregano essential oil, has been proposed as a potential inhibitor of biofilm formation and other virulence factors of many bacteria, including P. carotovorum (Burt et al., 2014, Joshi et al., 2016, Tapia-Rodriguez et al., 2017). Burt et al. (2014) reported that carvacrol (at subinhibitory concentrations, <0.5 mM) inhibited the biofilm formation of Chromobacterium violaceum, Salmonella Typhimurium and Staphylococcus aureus; whereas, Tapia-Rodriguez et al. (2017) observed that this compound (at 3.9 and 0.7 mM) caused a reduction of pyocianin and violacein, virulence factors regulated by QS system in Pseudomonas aeruginosa and C. violaceum, respectively. It has been hypothesized that the possible mechanism of action of carvacrol is through the interruption of QS mechanisms. Joshi et al. (2016) reported that after 24 h of exposure, carvacrol inhibited biofilm formation and reduced the synthesis of QS signal molecules in P. carotovorum subsp. brasilense. However, the effect of this terpene on motility, surface physicochemical properties (related with the initial adhesion of bacteria to food surfaces) and on-time EPS composition during the biofilm formation were not evaluated. In this sense, the purpose of this study was to characterize the effect of carvacrol on bacterial surface charge, adhesion potential, motility and on-time EPS composition during P. carotovorum biofilm formation.