Biofilm development on packaging materials

INTRODUCTION

The term ‘‘microbial biofilm’’ or ‘‘biofilm’’ refers to the complex aggregation of microorganisms growing on a solid substrate such as a packaging material, not a film that is made of biopolymer. This article provides an introductory understanding of biofilm and its formation. Since a major function of packaging is to protect the product from microbial contamination and its associated heath risks, an understanding of biofilm is important, particularly for products which require aseptic packaging and retortable packaging.

Bacteria on the surface develop a biofilm-associated community with higher resistance to toxic compounds (1, 2) than their planktonic counterparts in the bulk. In general, biofilms result from physicochemical conditions and interactions in the bacteria/environment complex (3, 4). A biofilm consists of a living microbial biomass surrounded by an exopolysaccharide envelope (EPS), proteins, and nucleic acids, which the biofilm microorganisms produce. These components help bacteria to attach to surfaces, stabilize local environment, and spatially organize communities that need to collaborate to use the substrate effectively (5). The process of the microorganism’s attachment to a surface is very complex, and the nature of both the microbial cell surface and the supporting surface (substratum) is critical for successful attachment (6). Surface adherence is an important survival mechanism for microorganisms. Moreover, the adhesion kinetics is the unique characteristic of a specific microorganism, differing even among phenotypes and strains (7). Several major factors affect attachment and consequently biofilm formation: the nature of the cell surface, the chemistry and texture of the attachment surface, the nature of the surrounding medium, and the temporal and spatial distribution of available nutrients (8–10).

A biofilm can be defined as a layer of microorganisms immobilized at a substratum held together in a multi-nature matrix polymer matrix (11). This matrix consists mainly of water (97%), microbial cells (2–5%), polysaccharides (neutral and polyanionic) (1–2%), proteins [including enzymes (1–2%)], and DNA and RNA from lysed cells (1–2%) (12). Usually, a mix consortium of microbes makes up this ecosystem, and they ‘‘team up’’ in order to protect themselves from stress and maximize nutrient uptake. One of the main components of a biofilm is the exopolysaccharide (EPS), which often consists of one or more family of different polysaccharides produced by at least some of the biofilm microorganisms. These components aid the attachment of cells to surfaces, stabilizing the local environment and spatial organization of the microbial communities, which may need to cooperate with each other to effectively use the available substrate (5).

BIOFILM ARCHITECTURE

A biofilm is a multiphase system. It consists of the biofilm itself, the overlying gas and/or liquid layer, and the substratum on which it (the biofilm) is immobilized. This system can be classified in terms of phases and compartments. The phases consist of the solid, liquid, and gas components, whereas the compartments consist of the substratum, the base film, the surface film, the bulk liquid and the gas. Biofilms are heterogeneous by nature, however, having stacks of cells scattered in a glycocalyx network with fluid-filled channels (13). Structurally, a biofilm is approximately two-dimensional, with its thickness ranging from a few micrometers to millimeters (5). This structure allows for the diffusion of nutrients and metabolic substances within the matrix.

Biofilm Organization.

The microbes in a biofilm are typically organized into microcolonies embedded in the EPS polymer matrix (5). These microcolonies attain distinct 2-D or 3-D structural patterns. Initially, microcolonies are separated by void spaces, but ultimately they merge into unique structures forming a mature biofilm. This spatial organization is very important to the biological activity of the biofilm.

Extrapolysaccharide (EPS).

EPS may vary in chemical and physical properties, but it is primarily composed of polysaccharides (11). Some of these polysaccharides can be neutral or polyanionic. Generally, this polymer can accommodate considerable amount of water into its structure by hydrogen bonding. Overall, the EPS has an important role of holding the biofilm together (14). As the EPS layer thickens, the biofilm microenvironment changes due to the activities of the bacteria. Therefore, a mature biofilm is a heterogeneous matrix. This heterogeneity concept is descriptive for both mixed and pure culture biofilms common on abiotic surfaces, including medical devices (15).

Quorum Sensing.

Quorum-sensing gene expression has been proposed as an essential component of biofilm physiology, since biofilm typically contains high concentration of cells (16). Generally, the irreversible attachment of bacteria to a substratum triggers alteration to an array of gene expression and phenotypes of these cells (17). In the quorum-sensing process, cell–cell communication is accomplished through the exchange of extracellular signaling molecules (16). For most gram-negative bacteria, the quorum-sensing regulation involves a freely diffusible auto-inducer, acylhomoserine lactone (AHL) signaling molecule. For instance, the quorum-sensing ability in P. aeruginosa is dependent upon two distinct but interrelated systems, las and rhl (18), which directs formation of the AHL. In gram-positive bacteria, structurally diverse peptides act as quorum sensing regulators (19). The QS system is not necessarily involved in the initial attachment and growth stages of biofilm formation but is very important in the overall biofilm differentiation process (20). During biofilm formation, QS signaling molecule mutants may develop thicker, more acid-resistant (21) or ‘‘abnormal’’ biofilms (22) than do the wild-type strains.

BIOFILM LIFE CYCLE

Once immobilized on a contact surface, microorganisms have the potential to form a biofilm. Attachment to the surface is beneficial to the microbe for a number of reasons. First, the surface represents important microbial habitats because in the microenvironment of a surface, nutrient levels may be much higher than they are in the bulk solution (23). Second, it increases the microbes’ resistance to mechanical and chemical stresses. Overall, biofilm formation is a dynamic process, comprised of four main stages (24): migration of cells to the substratum, adsorption of the cells to the substratum, growth and metabolic processes within the biofilm, and detachment of portions of the biofilm (see Figure 1). These steps can be divided into three phases: initial events, exponential accumulation and steady state.

Surface Conditioning Film Formation.

The conditioning film is created when organic materials (polysaccharides and proteins) settle on the surface (11). It can be derived from the microbes in the vessel or from the bulk fluid. Adsorption of a conditioning film is relatively quick compared to the other steps. This film has the potential to alter the physicochemical properties of the substratum and thus greatly impacts bacterial attachment.

Cell Migration to the Surface.

Migration of microorganism to the substratum is considered the second step in biofilm formation. This process can be mediated by different mechanisms depending on the system under consideration. Thus, transport can be (a) active, facilitated by flagella (25), or (b) passive, facilitated by Brownian diffusion, convection, or sedimentation. In quiescent systems (batch culture), sedimentation rates for bacteria are generally low due to their size and specific gravity (11), and microbes with a diameter of 1–4 mm3 have small Brownian diffusivity. Therefore, motility may be the limiting factor of transport in such systems. In a laminar flow system, although motility affects transport, diffusion remains the controlling factor. In a turbulent flow system, Brownian diffusion has minute contributions to transport, but forces such as frictional drag force, eddy diffusion, lift force, and turbulent bursts are significant.

Factors that Impact Interactions of Bacteria with a Substrate

Microbial adhesion is mediated by specific interactions between cell surface structures and specific molecular groups on the substratum. Moreover, the adhesion process is determined by physicochemical and molecular interactions. It is believed that primary adhesion between bacteria and abiotic surfaces is generally determined by nonspecific (e.g., hydrophobic) interactions, whereas adhesion to living or devitalized tissue is accomplished through specific molecular (lectin, ligand, or a adhesion) mechanisms (26).

Physicochemical Interactions.

Generally, two types of physicochemical interactions are used to describe the adhesion of a microorganism to a planar surface. The DLVO approach relates to the interaction energies (attraction and repulsion)—primarily to electrostatic and van der Waals forces—but chemical forces can operate (27). Typically, attraction between microbe and surface occurs either at a long range (5–8 nm), a secondary minimum, or at a shorter range, the primary minimum. Thus, adhesion can be reversible (at the secondary minimum) or irreversible (toward the primary minimum) (11). In the DLVO approach, the ionic charge of the medium, the physicochemistry of both the bacteria and substratum surface, and the physicochemistry of biosurfactant determine the extent of adhesion.

Alternatively, in the thermodynamic approach, adhesion is described as the formation of a new interface between the substratum surface and adhering bacteria at the expense of (a) the interfaces between bacteria and the suspending liquid and (b) the substratum–liquid interface (5). Each interface contains a specific amount of interfacial energy (or surface tension). The extent of adhesion is determined by the surface properties of all three phases, the surface tension of adhering particles, of the substratum and of the medium (28). The more hydrophilic a substrate, the higher is its surface tension.

Molecular Interactions.

Bacterial adherence is also mediated by molecular mechanisms. Bacteria are able to adhere to animal cells (29), such as muscle meats through protein–protein interactions on the surface. These proteins sometimes function as ligands to receptors when the bacteria invade target cells and/or have specific affinity for host components (30). The colony-opacity-associated (Opa) outer-membrane proteins or ligands (often called an a adhesion) confer intimate bacterial association with mammalian cells. Two classes of cellular receptors for Opa protein receptors have been identified: a adhesio-sulfate proteoglycan (HSPG) receptors and members of the carcinoembryonic antigen (CEA) or CD66 family (31). Listeria monocytogenes surface proteins Internalin A (InlA) and B (InlB) are involved in the attachment of this bacterium to host cells (32).

The magnitude of the cell substratum interaction forces, the chemical heterogeneity and the roughness of the substratum surface greatly affect the extent of microbial adhesion. As food traverses from the farm to the table, it comes in contact with fabricating equipments, utensils, gaskets, conveyor belts, packaging materials, storage containers, and chopping boards. These surfaces are usually metallic, plastic, rubber, or wood. Food processing equipments are often made of stainless steel, transport crates of high-density polyethylene (HPDE), conveyor belts of rubber, chopping boards of wood, and packaging materials of aluminum. Other storage and packaging-type materials include polypropylene, PVC, and Teflon. Sometimes the contact time between foods and surface may be 24–48 hours depending on the processing conditions including design of equipment cleaning and sanitation techniques.

Substratum Surface Hydrophobicity.

The hydrophobicity of the substratum has substantial effect on bacterial adhesion. Typically, hydrophilic surfaces such as stainless steel and glass have a high free surface energy and thus allow greater bacteria attachment than do hydrophobic surfaces such as Teflon (25). For instance, a general trend of decreasing colonization density was observed for Staphylococcus epidermis and Pseudomonas aeruginosa with an increase in substratum hydrophobicity (33). In the above-mentioned study, the packaging materials used were stainless steel, poly(vinyl chloride), polystyrene, and glass. Likewise, biofilm formation by L. monocytogenes LO28 was faster on hydrophilic (stainless steel) than on hydrophobic polytetrafluoroethylene (PTFE) (34).

Substratum-Surface Roughness/Topography.

Many reports have indicated that metal surfaces with a high degree of roughness serve as a better substrate for bacterial attachment than do smooth ones, since the surface area of the former is greater (33, 35). Arnold and others discovered that resistance to bacteria attachment decreased in the following order: Electropolished > Sanded > Blasted > Untreated Stainless Steel. Bower and Daeschel (1) illustrated that surface topography is extremely important in biofilm formation and resistance. Still studies such as that of Barnes et al. (36) claimed that the difference in bacteria attachment due to difference in surface topography is minimal.

Substratum Coverage with Organic Material.

The layer of organic substances present on the surface can be favorable or unfavorable to bacteria adhesion. Barnes et al. (36) discovered that proteins that adsorbed to a stainless steel surface inhibited bacterial attachment. The dominating mechanism was suspected to be competitive inhibition, since the proteins were able to interact with the hydrophilic surface. The adhesion process begins, provided that the conditioning film is favorable to bacterial attachment.

Bulk Nutrient Composition.

Generally, bulk fluid conditions influence surface hydrophobicity, adhesion expressions, and other factors that affect adhesiveness (37). Microbes are usually exposed to a range of nutrients concentrations from as low as 1 mg/L to 500 g/L, and this range has an effect on biofilm growth (5). At the highest nutrient concentrations, biofilms can appear to be uniform with few or no pores. This is common of biofilms associated with animal and food surfaces. Various reports suggest that the lower the concentration of nutrients, the greater the rate of attachment and biofilm development (38–40). Escherichia coli O157:H7 biofilms developed in minimal salts medium (MSM) developed faster and had thicker extracellular matrix, and cells detached much slower compared to those grown in trypticase soy broth (TSB) (38).

Temperature.

Temperature effect is particularly important in the food industry, since food will experience differentials in temperature (temperature abuse) from farm to fork. This abuse is a consequence of changes in cell wall and attachment factors (41). Stopforth et al. (42) showed that a greater number of Listeria monocytogenes cells adhered to stainless steel templates at 59 1F and 77 1F compared to 41 1F and 95 1F. Moreover, Stepanovic et al. (43) suggested that a microaerophilic environment supports biofilm formation. Presently, there are few conclusive reports that support this claim.

Ionic Strength.

The atomic ions present in the medium can indirectly affect attachment of the bacteria to other substratum. These ions may act as chelator, forming bridges between protein molecules on the bacteria surface and adsorbed proteins on the substratum surface. For example, with milk-treated steel, ferrous ions in solution increased Listeria monocytogenes attachment (36). Ions in the solution can also act as shields, shielding the surface charge of the substratum and the bacteria (44) and increasing bacteria attachment (45, 46). The ionic composition in the bulk may affect the composition of the metabolic by products of biofilm cells but not necessarily affect the physical property of the biofilm (47).

Hydrodynamics.

The flow velocity in close proximity to the substratum and the liquid boundary (hydrodynamics) has marked influence on the cellular interaction and the biofilm structure. Cells behave as particles in a liquid, and the rate of settling and association with a submerged surface depends greatly on the velocity characteristics of the liquid (48). After the bacteria has attached, flow rate or shear force of the liquid affects the biofilm structure and content (25). More compact, stable, and denser biofilms were formed at relatively higher hydrodynamic shear force (49).

Atmosphere.

The incubation atmosphere also influences biofilm formation. For instance, a microaerophilic and carbon dioxide-rich environment provided a relatively high rate of biofilm formation, whereas the least amount of biofilm was formed under anaerobic conditions (43). On the other hand, anaerobic growth favors maintenance of mucoid alginate (polysaccharide) production by Pseudomonas in cystic fibrosis airways (50).

BIOFILM FORMATION ON THE SURFACE OF THE PACKAGING MATERIALS

All ‘‘real-life’’ surfaces have substantial nonuniformity, with the surface irregularities (patterns) size ranging from nanometers to hundreds of microns. Although a number of studies have investigated the influence of the surface topography on biofilm formation by various microorganisms, including foodborne pathogens (1, 10, 51–53), most published results are devoted to the biofilm development in flow-through systems. It is difficult to separate the effects of surface patterns and those of the liquid flow on bacteria adhesion in such systems.

An ability to adhere to a surface provides an important survival mechanism for microorganisms (9). The process of microorganism’s attachment to the material surface is very complex, and the nature of both the microbial cell surface and the supporting substrate is important (6). For example, an electropolished stainless steel substratum showed significantly lower bacterial cells adhesion rate and delay in biofilm formation, compared with the sandblasted one (54). Planktonic microbial cells are delivered by diffusion and motility from a bulk medium to the surface, where a fraction of those cells adheres to the surface. The dynamics of bacterial adhesion is a unique characteristic of the specific microorganism; there may even be differences among the phenotypes and strains of the same bacterium (7).

Bacterial colonization of surfaces is influenced by two factors: First, a well-developed surface has higher adsorption capacity, and therefore the preconditioning organic film necessary for bacteria attachment is more likely to be formed on such a surface. On the other hand, surface topography influences bacterial attachment and proliferation, limiting the directions of colony growth and limiting nutrient access.

Surface topography was found to greatly affect the behavior and morphology of bacterial cells within colonies during the initial stages of biofilm development. In an effort to maximize their survival rate, bacteria form clusters of unique shapes, ranging from two-dimensional (2-D) single-layer colonies to three-dimensional (3-D) pillar- like structures within grooves. Hence, it is possible to control initial colony shape by varying the characteristics of surface constraints. Coupled with surface topography, starvation may play an important role in the attachment of bacteria to the surface, which is directly supported by our observations of bacterial behavior in the surface confines with limited nutrient access.

In general, surface patterns (i.e., roughness) impact microbial population in several ways: A well-developed (patterned) surface has higher adsorption capacity, whereas the presence of highly inclined regions (constraints) makes cell attachment more difficult. As follows from the obtained data, initial biofilm formation on rough surfaces occurs in two dimensions. If nutrient access is limited by the configuration of surface constraints and/or diffusion transport, bacteria can develop 3-D structures. On the other hand, if the tested surface is plain and smooth, bacteria always spread over it as a single-layer (2-D) colony. Maturing of the biofilm and corresponding total surface coverage lead to the development of threedimensional structures, which have been previously described in the literature (4, 55, 56) and also observed in our experiments.

Kinetics of bacterial adhesion onto surface of selected packaging materials is depicted in Figure 2. These results support data in the literature that adhesion of Listeria monocytogenes is material-dependent (34, 57).

FOODBORNE BIOFILMS AND ACTIVE (CONTROLLEDRELEASE) PACKAGING MATERIALS

The food market has growing demand on fresh and minimally processed foods. However, these foods are highly perishable and more susceptible to microbial spoilage. Thus, there is a strong need to develop new preservation methods to achieve a required level of safety, quality, and nutritional value of food during extended shelf-life period. The use of active packaging (AP) materials is one of the post-processing methods to preserve food products and meet consumers’ expectations.

Antimicrobial packaging is designed to control microbial growth in a food product. It consists of an antimicrobial agent (AMA) immobilized onto the internal surface of a package or incorporated into packaging material (58). In the latter case, AMA is released into a food product over time. This permits us to extend shelf life of food products, helping to reduce the amount of AMA in food formulation.

AP materials have many parameters that influence their antimicrobial efficacy, which is thoroughly addressed in a number of studies: polymer processing, polymer morphology (59), polymer swelling (60), and AMA affinity to the packaging material (61). In all these papers the food product was considered as a homogeneous medium in full contact with the packaging. However, surface morphology of the foods is an important parameter that determines mass transfer of an antimicrobial agent through the interface between packaging and food.

Based on the nature of a food product and corresponding morphology of a food surface, one can distinguish five types of food-packaging contacts depicted in Figure 3.

If the surface of a food product is flat, direct contact between the packaging and the food exists. This AP system has maximum efficacy. An irregular food surface will cause only partial contact between the packaging material and the food product, developing noncontinuous headspace. This influences AMA transport from the packaging to the food. Depending on the dominating physical state of a food product, the packaging surface can be in contact with either solid or liquid food products, sometimes both. These contacts can be direct or indirect if headspace exists between the food surface and packaging material (see Figure 4). Depending on the type of the food product, the headspace can be liquid- or gas-filled.

Numerous parameters can influence the efficacy of AP, including the packaging material properties, the antimicrobial transport, the bacterial population response and the food matrix. Most of the published studies investigated antimicrobial activity of the controlled release compound by adding AMA directly to the foods or to the packaging materials. However, these two methods have significant disadvantages:

• When AMA is added directly to the food, obtained data provide important information on the antimicrobial activity of AMA and its interaction with the food matrix. But there is no time-dependent AMA release; therefore these studies are insufficient for the development of AP. 
• On the other hand, if AMA is incorporated into the packaging material, there is no control over the antimicrobial release. The effects of packaging material properties on the AMA release rate cannot be distinguished from the effects of the AMA release rate on the bacterial inhibition. 

There is a need to understand bacterial response to the AMA release without the influence of material-dependent properties of AP. No standard method has been established to investigate the effect of antimicrobial agent’s timedependent release on the bacterial response.