Controlling Biofilm Formation of Foodborne Pathogens Utilizing Probiotics in the Food Industry

INTRODUCTION

Foodborne illnesses continue to pose a serious global threat to public health, contributing significantly to morbidity, mortality, and economic losses across the world [1]. A major challenge in controlling these infections is the way foodborne pathogens build biofilms. In food processing settings, biofilms—structured colonies of microbial cells encased in a self-made polymeric matrix—stick to surfaces like rubber, plastic, and stainless steel [2]. Once formed, these biofilms give bacteria a safe haven that increases their resilience to environmental stressors, disinfectants, and antimicrobial treatments. This raises the possibility of ongoing contamination in systems used to produce food [3, 4].

The contamination of food products can occur at various stages along the food supply chain—including production, processing, packaging, transportation, and final preparation—making biofilm control a critical component of food safety management [5]. Not only do biofilms act as reservoirs for pathogens, but they also lead to sensory and quality degradation in food, affecting taste, smell, texture, and shelf life [6].

Common biofilm-forming foodborne pathogens include Salmonella spp., Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, Clostridium perfringens, and Vibrio spp., all of which have been linked to significant outbreaks and food recalls globally [7]. According to the European Food Safety Authority (EFSA), Salmonella and Campylobacter were the most frequently reported causative agents of zoonotic foodborne diseases in 2020, followed by Listeria and Yersinia [8].

The food industry has used a variety of techniques, including mechanical scrubbing, thermal processing, chemical sanitisers, and surface coatings, to reduce the production of biofilms.  However, because of the tenacity of bacteria buried in biofilms and the possible hazards to the environment and human health posed by chemical treatments, these methods frequently fail [9, 10]. Consequently, the search for safer, sustainable, and more effective alternatives has gained momentum.

One promising approach is the use of probiotics—live microorganisms that confer health benefits when administered in adequate amounts [11]. Beyond their traditional role in gut health, probiotics have demonstrated potential in inhibiting biofilm formation by foodborne pathogens through various mechanisms, including competitive exclusion, production of bacteriocins and organic acids, co-aggregation with pathogens, and disruption of quorum sensing (QS) pathways [12-14]. Recent studies have shown that strains such as Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, and Pediococcus acidilactici exhibit strong anti-biofilm properties in in vitro and situ models [15, 16].

Moreover, the concept of probiotic cocktails, combining multiple strains, has emerged as a more robust strategy for biofilm disruption, offering synergistic effects and broader antimicrobial spectra [17]. Despite the promising findings, there remains a need to explore the practical application of probiotics in food processing environments, the stability of their anti-biofilm activity under industrial conditions, and their regulatory acceptance as part of food safety interventions.

The purpose of this narrative review is to examine the current understanding of foodborne pathogen biofilm formation and the growing role of probiotics as an all-natural and successful biofilm management method in the food business.

MATERIALS AND METHODS

Literature Selection Criteria

A comprehensive search of the available literature was conducted using several electronic databases, including PubMed, Scopus, and Web of Science. Studies published between 2000 and 2024 were considered for inclusion. The selection process involved the following inclusion criteria:

• Studies focused on foodborne pathogens and probiotic interventions in the food industry.
• Peer-reviewed articles, clinical trials, experimental studies, and systematic reviews.
• Articles available in English.

Exclusion criteria included:

• Studies not related to probiotics or foodborne pathogens.
• Non-peer-reviewed sources (e.g., conference abstracts, editorials).
• Studies focusing on non-food-related applications of probiotics.

The final selection was based on the relevance and methodological quality of the studies, ensuring a comprehensive representation of the current state of research.

RESULTS AND DISCUSSION

• Biofilm Formation

Biofilms are cohesive structures composed of microorganisms that adhere to biotic and abiotic surfaces. Microbes wrap themselves in an extracellular matrix through the secretion of extracellular polymers. Essentially, the outer layers of microbes connect with the surfaces of intricate groups of microbes. Bacteria generate a polymer matrix composed primarily of biomolecules. This polymer matrix creates a well-aerated and moisturized combination which plays a crucial role in maintaining the biofilms and their 3-D (dimensional) configurations. Significantly, the features of biofilms offer microorganisms safeguard from natural elements along with the increase of the defense against antibacterial treatments, hence helping with the endurance and harmfulness of microbes. Therefore, the production of bacterial biofilms is a crucial component of the bacterium's mechanism for survival [10]. The morphological composition and susceptibility to ecological influences along with living attributes of microbes in biofilms differ significantly from the microbes in plankton. Additionally, the 3-D arrangement that biofilms have served as an inherent defense along with a shielding coating against microbes [11].

The development and growth processes of biofilms are continuing, ever-changing, and intricate procedures that work based on factors such as the matrix, culture media, essential cell properties, messenger molecules, cellular biomolecule processes, and hereditary regulation. The process of biofilm development comprises five consecutive stages and these stages are shown in Figure 1:

This figure illustrates the sequential stages of biofilm development: initial attachment, irreversible attachment, maturation I and II, and dispersion. It highlights how free-floating (planktonic) bacteria adhere to surfaces, form microcolonies, develop complex extracellular matrices, and eventually disperse to colonize new sites. Understanding these stages is critical for identifying points of intervention to disrupt biofilm formation [12].

In order to create an appropriate surface layer, bacterial biofilms begin with the absorption of either organic (like proteins, lipids, polysaccharides, fatty acids, etc.) or inorganic (like inorganic salt, water, etc.) molecules.  Following that, this layer is integrated into a variety of Extracellular Polymeric Substances (EPS) in either single or mixed communities [13]. After bacteria have adhered to biotic and abiotic outer layer, they engage in intercellular communication using an extracellular signaling mechanism known as QS. By encouraging particular genetic material in microbes to produce extracellular matrix, including EPS and proteins, QS regulates the entire biofilm growth process, leading to the gradual development of a fully developed and mature biofilm structure.  QS's ability to communicate between cells is crucial to the formation of biofilms.  Bacterial cells use a process called QS to control the production, release, and buildup of signal molecules outside of their cells through chemical communication [14].

Role of Biofilm in Food Industry

Inter-mixing of pollutants in food may occur at any point in the food supply progression, including manufacturing, procedure of processing, preservation, dissemination, and preparation of food by customers [3]. Food contamination can result in alterations in flavor, fragrance, surface quality, and visual aspects that are regarded as unsatisfactory and unwanted [4]. Raw, uncooked, little processed food, primarily derived from animals but also including fruits and vegetables, is very susceptible to bacterial contamination [13]. Bacteria tend to attach themselves to surfaces that come into touch with food and create biofilms, rather than remaining in a free-floating state in the water. Biofilms present a significant hygiene threat as they serve as reservoirs for foodborne pathogens. These microbes modify the sensory food qualities by releasing lipases and proteases [15]. The presence of nutrients along with dampness on the outer layers facilitates the growth of biofilms that occur on many hard materials such as fruits, meats, bones, and food industry equipment made of stainless steel, plastic, polystyrene, and glass [16].

Listeria monocytogenes is a major foodborne pathogen that forms biofilm when in contact with hard surfaces like stainless steel, plastics, etc. Food is contaminated when comes into contact with such hard surfaces [17].

It has been discovered that slaughter areas, such as equipment used in chicken processing, can produce isolates of Salmonella. Because the environment is typically damp, biofilm development is highly favorable. Though the prevalence of Salmonella biofilms in food processing environments is poorly understood, research has demonstrated and confirmed Salmonella's ability to attach to and form biofilms on surfaces such as those found in food processing plants' stainless steel, cement, and plastic [17].

Foodborne diseases are caused by using items like milk and other dairy products and are caused by Staphylococcus aureus, a significant foodborne pathogen with the ability to form biofilms. In the dairy sector, biofilm formation often happens on almost every surface of technological systems [17].

• Pathogenic and Foodborne Microbes

In 2020, Salmonella spp. was responsible for the most reported cases of foodborne illnesses, followed by Listeria monocytogenes. Other important bacteria that cause foodborne diseases include Vibrio spp., Clostridium spp., Staphylococcus spp., and Pseudomonas aeruginosa [5]. Symptoms of Salmonella include fever, diarrhoea, and gastrointestinal distress. Acute gastroenteritis and more serious conditions including meningitis and abortion can be brought on by L. monocytogenes. Acute gastroenteritis can be brought on by S. aureus [18].

The pathogenic microbes that cause foodborne diseases are given in Table 1 with their source of infection and symptoms caused by them.

• Methodology to Manage and Inhibit Biofilm Development

The best way to get rid of biofilms in the food business is to actively prevent them from growing and, as important, prevent bacteria from entering food processing facilities. Implementing an efficient hygiene procedure and ensuring the design of the plant and equipment is essential to restrict the entry of microbes into industries and prevent their interaction with food [15, 33]. To reduce areas where microorganisms can take shelter and proliferate, it is advisable to avoid gaps and cracks [15]. The selection of exterior layer materials and coverings is crucial in preventing the growth of biofilms [33]. Furthermore, the implementation of a Hazard Analysis and Critical Control Point system (HACCP) is crucial to maintaining the well-being and standard of food [34]. The methods to regulate and avoid biofilm formation are shown in Figure 2 in a flow diagram.

There are a few methods that help to regulate and avoid biofilm development, and these are described in this paragraph. After biofilms have developed in food contact areas, the initial methods used to remove them are Mechanical and Physical Cleaning techniques [33]. These techniques include high-pressure cleaning and the injection of extremely hot steam [16]. The biofilm is destroyed as a result of these activities interfering with the extracellular matrix [8]. One method for managing biofilm is chemical treatment with detergents, sanitizers, and decontaminants [35, 36]. It has been demonstrated that chemical treatments such sodium hydroxide, hydrogen peroxide, peracetic acid, and sodium hypochlorite can decrease biofilms [6]. Environmentally friendly methods for controlling biofilm include employing enzymes, bacteriophages, natural substances like concentrated plant oils, and chemicals produced by bacteria, for instance, bacteriocins and biosurfactants [36]. Enzymes, such as proteases, lipases, and polysaccharides, are large as well as biodegradable biologically active molecules that can prevent biofilm formation. Bacteriophages are viruses that specifically infect prokaryotic cells. Essential oils are composed of a combination of secondary metabolites derived from plants, such as phenol, thymol, and carvacrol [8]. QS inhibition is seen as an alternative method to control biofilm formation, however, the exact connection between the two is not yet completely comprehended [6]. Microorganisms produce quorum-quenching chemicals as a means of competing with nearby cells [13].

All these methods discussed above have some disadvantages which make them unable to be used in certain cases. The properties of the surface being cleaned, the type of biofilm, the strength of the cleaning agents, and the length of time and temperature of the CIP (Clean-in-Place) process all affect how effective the cleaning process is [37]. In the case of Chemical treatment, the dosage and the timing concerning these chemical products are typically adjusted to eradicate aquatic plankton bacteria, making them potentially ineffective against biofilms [34]. In addition, biofilms exhibit greater resistance to certain biocidal agents, such as chlorine-containing and quaternary ammonium sanitizers [6].  While dealing with Environmentally friendly methods the bacteriophages have shortcomings in effectively directing against microbes within biofilms because of the presence of the extracellular matrix. Some of the essential oils may irritate the dermis and internal human system [6]. QS inhibition can occur via a number of methods. Autoinducers (AI) are QS signaling molecules that inhibitors can bind to competitively, quorum-quenching enzymes can degrade AI signals, small regulatory RNAs (sRNAs) can post-transcriptionally modulate QS genes, and AI can be directly blocked. QS genes can be inhibited and the QS mechanism suppressed by interfering with just one element of the QS pathway [38]. All conventional methods to control biofilm formation have certain limitations. Probiotics have therefore become a viable substitute tactic to prevent biofilm formation in the food sector, lowering the possibility of antibiotic resistance linked to foodborne infections [5].

When taken in sufficient quantities, probiotics—live microorganisms commonly known as "good bacteria"—produce health advantages. They are frequently present in cultured milk products and fermented foods. Probiotics can be found in abundance in these fermented foods.

Although probiotics can be derived from a range of microorganisms including bacteria, yeasts, and molds, the most widely used types belong to the bacterial genera Lactobacillus, Lactococcus, Streptococcus, and Bifidobacterium. Probiotics are known to help mitigate adverse health effects and support overall well-being [39].

Several studies have shown that some probiotics, especially lactic acid bacteria (LAB), can control the production of biofilms by different pathogens and prevent microbial cell adhesion. Figure 3 provides an illustration of this method [40, 41].

The competition for resources and binding sites, as well as the release of antimicrobial compounds produced from microorganisms, such as bacteriocins, biosurfactants, organic acids, hydrogen peroxide, and restricted exopolysaccharides, may all contribute to the antagonistic effect [42]. Furthermore, earlier studies have shown that probiotics improve food safety by preventing QS activity [38]. Probiotics work by using Bacteriocins to undermine the integrity of bacterial cells. This is accomplished by releasing the proton motive force and either preventing the formation of peptidoglycans or by creating pores in bacterial membranes.  Lactic acids and other organic acids lower pH, which may prevent microorganisms from growing while having no effect on probiotics because they can tolerate low pH levels [42]. Foodborne viruses can enhance their existence in the gastrointestinal tract by creating biofilms after they enter the human body. The production of these biofilms is controlled by a process called QS. Therefore, probiotics, which are consumed through fermentation-based foods, have a twofold impact on both the well-being and standard of food, as well as on gut health. This is potentially achieved by interfering with the QS activity of harmful bacteria [38].

Methods Using Probiotics to Inhibit Biofilm Development

Probiotics can suppress the proliferation of microorganisms and prevent biofilm development using certain methods which are displacement, exclusion, or competition, and are displayed in Figure 4 in a Flow Diagram [9].

This flow diagram categorizes the primary probiotic strategies for inhibiting biofilm formation into three core mechanisms: displacement, exclusion, and competition.

Displacement

Displacement involves the introduction of probiotics and/or their metabolites to disturb pre-existing biofilms [9].

Probiotics such as Lactobacillus and Bifidobacterium species can compete with pathogenic bacteria for attachment sites on surfaces. This competition results in the displacement of pathogenic bacteria and the prevention of their colonization [43]. 

Exclusion

Exclusion involves applying probiotic biofilms and/or their metabolites onto food contact surfaces to hinder the attachment of pathogenic bacteria [44].

Exclusion refers to the targeted prevention of the formation of harmful biofilms by probiotic strains through mechanisms such as the generation of antimicrobial compounds and competition for resources. Probiotics can generate bacteriocins, organic acids, and other antimicrobial substances that hinder the growth and formation of biofilms by harmful bacteria such as Salmonella and Listeria [43].

Competition

The competition includes the first-hand contact between probiotics and/or their metabolites and microbes present in food that can cause illness [9].

Probiotics can disturb the microbial balance, prevent pathogenic colonization, and reduce biofilm growth in different situations by actively competing with pathogens for resources necessary for biofilm formation [43].

Outcomes Obtained by Performing the Displacement Method

Research regarding utilizing probiotics to manage and block the production of biofilms within the food sector has been growing. Different probiotic genera are commonly used. The prevailing techniques employed for biofilm evaluation include crystal violet (CV) staining and colony forming units (CFU) counting. Multiple materials have been investigated for the production of biofilms, with glass and polystyrene being the most commonly used followed by stainless steel. Wood, rubber, silicone, polyvinyl chloride, and polytetrafluoroethylene were also examined. Microtiter plates served as the main platform for biofilm formation in the bulk of the studies. Of the anti-biofilm compounds that were analysed, probiotic cells and cell-free supernatant (CFS) were the most often investigated, making up 34% and 31% of the research, respectively. To a lesser degree, EPS, biosurfactants, crude extracts, and bacteriocins were also investigated. Table 2 illustrates how probiotics can use the displacement approach to regulate the biofilms that form on meals. The following probiotics are used to regulate the production of biofilms: