# Biofilm Formation in Dairy: A Food Safety Concern-Recent progress in antibiofilm strategies in the dairy industry

Coralie Goetz, Laurie Sanschagrin, Eric Jubinville, Mario Jacques, Julie Jean

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Coralie Goetz, Laurie Sanschagrin, Eric Jubinville, Mario Jacques, Julie Jean. Biofilm Formation in Dairy: A Food Safety Concern-Recent progress in antibiofilm strategies in the dairy industry. Journal of Dairy Science, 2025, 108 (8), pp.8157-8175. 10.3168/jds.2024-25554. hal-05491063

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# Biofilm Formation in Dairy: A Food Safety Concern—Recent progress in antibiofilm strategies in the dairy industry

Coralie Goetz,<fnref n="1" />* Laurie Sanschagrin,<fnref n="2" />* Eric Jubinville,<fnref n="2" /> Mario Jacques,<fnref n="3" /> and Julie Jean<fnref n="2" />†

## ABSTRACT

Biofilm formation allows microorganisms including bacteria to persist on abiotic or biotic surfaces, to resist treatments with biocides (disinfectants and antibiotics), and to evade the immune response in animal hosts much more than they do in the planktonic form. Bacteria able to form biofilm can be troublesome in the dairy industry, both by causing clinical symptoms in livestock and by colonizing milking devices and milk processing equipment, resulting in dairy products of lower quality and sometimes raising serious food safety issues. In fact, most of the bacterial species isolated frequently in the dairy chain have the ability to form biofilm. Common examples include *Staphylococcus aureus* and other staphylococci that frequently infect mammary glands but also *Bacillus* spp., *Listeria monocytogenes*, and *Pseudomonas* spp., which cause spoilage of dairy products and sometimes foodborne illnesses. The economic losses due to biofilm formation in the dairy industry are considerable, and scientists are constantly solicited to develop new antibiofilm strategies, especially using biocides of natural origin. Although the number of studies in this subject area has exploded in recent years, the in vivo efficacy of most novel approaches remains to be explored. Used alone or to increase the efficacy of disinfectants or antibiotics, they could allow the implementation of strategies having less impact on the environment. Their use is expected to lead to less reliance on antibiotics to treat intramammary infections in dairy farms and the use of lower concentrations of chemical disinfectants in dairy processing plants.

**Key words:** dairy industry, bacteria, biofilm, antibiofilm, biocide

## INTRODUCTION

Despite implementation of very strict hygienic measures from the dairy farm to the dairy processing plant, the risk of contamination by microorganisms including bacteria remains omnipresent throughout the dairy chain (Montgomery et al., 2020). Most of these microorganisms cause mainly spoilage of products, but some are pathogens and can cause diseases in livestock and illness in consumers (Montgomery et al., 2020). Of particular concern is their ability in certain cases to adhere to dairy industry surfaces, biotic or abiotic, to form biofilm, a community of cells aggregated into an extended colony covered with a polymer matrix (Melchior, 2011; Marchand et al., 2012).

In dairy farms, biofilm-forming bacteria are often associated with bovine mastitis, an inflammatory infection having a major negative influence on animal well-being, dairy yield, and milk quality (Pedersen et al., 2021; Morales-Ubaldo et al., 2023). The biofilm-forming ability of several mastitis pathogens, including *Staphylococcus aureus*, CNS, *Escherichia coli*, and *Streptococcus uberis*, has been demonstrated in vitro using conventional methods, such as 96-well microtiter plates (Melchior, 2011; Tremblay et al., 2013). Furthermore, in vivo studies highlighted the presence of bacterial biofilms in the bovine udder (Hensen et al., 2000; Schönborn and Krömker, 2016). The latter could explain the failure of certain antibiotic treatments to cure bovine mastitis, supporting the need for alternative strategies in dairy herds (Ceri et al., 2010; Morales-Ubaldo et al., 2023).

In dairy processing plants, biofilms are thus a threat both to the economic viability of dairy processing plants and human health. Indeed, biofilm-forming microorganisms will have a negative effect on product quality and safety as a result of constant shedding of microbial cells from biofilm into milk, before and after pasteurization (Marchand et al., 2012; Chamberland et al., 2017; Sanschagrin et al., 2024). These contaminants include *Bacillus* spp., *Enterobacter* spp., *Klebsiella* spp., *Listeria monocytogenes*, *Pseudomonas* spp., *Staph. aureus* and

<footnote n="1">¹INRAE, UMR1253 STLO, L'Institut Agro Rennes-Angers, 35042 Rennes Cedex, France</footnote>

<footnote n="2">²Département des Sciences des Aliments, Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Québec, QC G1V 0A6, Canada</footnote>

<footnote n="3">³Regroupement de Recherche pour un Lait de Qualité Optimale (Op+Lait), Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, QC J2S 2M2, Canada</footnote>
*Streptococcus* spp. Their biofilms are difficult to elimi-
nate using standard clean-in-place (CIP) procedures and
can become resistant to sanitizing agents (Marchand et
al., 2012; Teh et al., 2015). It may become necessary to
implement an additional wash cycle, and thus accept a
loss of productive hours or the cost of extra hours. Bio-
films also favor corrosion of stainless steel surfaces and
lead to earlier replacement of processing equipment, pip-
ing, and storage tanks (Friedlander et al., 2019). The eco-
nomic losses attributable to biofilms are considerable for
the dairy industry from the farm to the processing plant
(Marchand et al., 2012; Morales-Ubaldo et al., 2023).
Furthermore, the presence of biofilm-forming bacteria in
the dairy chain may be a cause of failure of treatments
with biocides, and thus contribute to the dissemination
of resistance. Dairy farmers and processors must face
this latent threat and evolve their practices toward more
sustainable approaches that respect the environment, ani-
mals, and humans. Researchers are therefore searching
to optimize or to develop novel and more effective ap-
proaches to prevent bacterial adhesion, and thus biofilm
formation. Knowledge of the components and the various
stages of formation of biofilm has suggested 3 strategic
options in human medicine (Chung and Toh, 2014): (1)
inhibiting the adhesion of planktonic bacteria to surfaces
to stop biofilm formation before it starts; (2) disrupting
biofilm architecture during the maturation phase; and (3)
interfering with intracellular signals within the biofilm.
The use of compounds exerting at least one of these ef-
fects in the dairy industry should allow us to reduce the
use of biocides and limit the spread of biocide resistance
and residues. The purpose of this review is to summa-
rize the current knowledge on the problems caused by
biofilm-forming bacteria in the dairy industry, from farm
to processing plants, and to discuss the various strategies
proposed to prevent their formation or eliminate them
once formed. This work also highlights the motivation to
investigate alternatives that are more respectful to the en-
vironment, animal health and welfare, and human health,
as part of a One Health approach.

BACTERIAL BIOFILM

Generalities

A bacterial biofilm is a community of cells aggregated
into an extended colony covered with a polymer matrix
that helps it adhere to a surface, which may be biotic
(for example, mucous membranes of an animal host) or
abiotic (for example, milking equipment on a dairy farm;
Costerton et al., 1999; Jacques et al., 2010). This mode of
life allows the bacteria to colonize environments that they
otherwise would not by affording them greater resistance
to cleaning agents, disinfectants, antibiotics, and the host

immune response than they have in the planktonic form
(Jacques et al., 2010; Hathroubi et al., 2017).

It has been proposed in the more recent history of
biofilm studies that an intermediate bacterial “life-
style” may be common, that of bacterial aggregates or
fragmented biofilm in suspension (Cai, 2020). Medical
literature in particular contains reports of bacterial ag-
gregates that are not attached directly to any surface
(Høiby et al., 2015; Kragh et al., 2016, 2023). Studies of
chronic infections in humans have revealed that such ag-
gregates range from about 5 µm to 200 mm in diameter
(Bjarnsholt et al., 2013). They have also been observed
in association with pig lung infections by *Actinoba-*
*cillus pleuropneumoniae* (Tremblay et al., 2017) and
cow mammary gland infections with *Staph. aureus* (I.
Doghri, M. Jacques, S. Nichols, J.-P. Roy, and S. Dufour,
Université de Montréal, St-Hyacinthe, QC, Canada, per-
sonal communication). Studies of *Salmonella* spp. show
that biofilm and aggregates are not mutually exclusive
(MacKenzie et al., 2015).

Whereas formation of biofilm or aggregates seems
necessary for the persistence of some bacterial species in
their hosts or environment, in other cases, such forma-
tions might play an important role in their persistence
in ecological niches, such as water, soil, surfaces inside
farm buildings and processing plants, or in transmission
between individuals or between animals and humans
(Jacques and Malouin, 2022). In vitro studies have fo-
cused mainly on “single-species” biofilms, even though
it has been clear that “multispecies” biofilms are the
rule rather than the exception in animal hosts and under
most environmental conditions (Elias and Banin, 2012;
Burmølle et al., 2014; Røder et al., 2020). Interactions be-
tween species influence the development and shape of the
community (Elias and Banin, 2012). In general, interspe-
cies interactions involve communication and metabolic
cooperation or competition (Elias and Banin, 2012).

Common Characteristics of Biofilms

Bacteria that are able to form biofilm have certain char-
acteristics in common (Costerton et al., 1999; Tremblay
et al., 2014). To begin with, the cells are encased in a
highly hydrated polymer matrix (>90% water) composed
of secreted exopolysaccharide, protein, and nucleic ac-
ids, albeit in widely varying proportions. For example,
*Staph. aureus* intercellular adhesin consists almost en-
tirely of poly N-acetyl-D-glucosamine (Arciola et al.,
2015). Second, biofilm development is a response to ex-
tracellular signals from the immediate surroundings or to
quorum sensing (QS) or similar gene-expression-based
regulation by the bacterial cells themselves (Miller and
Bassler, 2001). Quorum sensing is a mechanism whereby
bacteria sense their numbers within a given space, based
<figure><img src="image_3.png" /><figcaption>**Figure 1.** The stages of biofilm formation: (1) aggregation and attachment, during which planktonic bacteria form suspended colonies (aggregates) or attach directly to a substrate (surface), which may be biotic or abiotic; (2) growth and accumulation, by cell division and recruitment of suspended cells or cell masses from the surrounding medium; and (3) disaggregation and detachment, dispersion of biofilm fragments and single cells (Sauer et al., 2022).</figcaption></figure>

on the concentrations of certain signal molecules that activate transcriptional regulators. It determines biofilm composition and thickness and initiates the dispersion phase (Pan and Ren, 2009). Third, biofilm protects the cells against harm by the host immune system, disinfectants, antibiotics, and desiccation (Costerton et al., 1999; Hall-Stoodley and Stoodley, 2009). It is interesting that aggregation seems to impart to bacteria most of the benefits of biofilm, including greater resistance or tolerance to antimicrobial agents (Kragh et al., 2016).

## The Different Stages of Biofilm Formation

For many years, biofilm formation was described in the literature using a 5-phase model: (1) development of a conditioning film on a surface; (2) congregation of planktonic bacteria on that surface; (3) adhesion (reversible then irreversible) of bacterial cells to the surface; (4) microbial cell growth and division then formation of microcolonies and maturation of the biofilm; and (5) dispersion of cells from the biofilm (Sauer et al., 2002). To take into consideration bacterial aggregates not attached to a surface, a revised model comprising 3 main phases has been recently proposed: (1) aggregation; (2) growth; and (3) disaggregation in a cycle (Figure 1; Sauer et al., 2022). This revised conceptual model encompasses the 3 major steps of biofilm life, aggregation, growth and disaggregation independently of surfaces, and initiation from single-cell planktonic bacteria, and is flexible enough to include all the diverse scenarios and microenvironments where biofilms are formed (Sauer et al., 2022).

To study biofilm formation and dispersion, and to determine the efficacy of compounds proposed as antibiofilm agents, a wide variety of laboratory techniques are available (Castelo-Branco et al., 2020). Among these are static methods, such as tests in 96-well microtiter plates (Christensen et al., 1985), the “Calgary Biofilm Device” or minimal biofilm eradication concentration assay (Ceri et al., 1999), and the “biofilm ring test” (Chavant et al., 2007), and dynamic methods, such as the “drip flow cell” (Goeres et al., 2009), continuous flow devices (Benoit et al., 2010), and the “CDC biofilm reactor” (Niboucha et al., 2022), as well as combined static and dynamic methods (Goetz et al., 2022b).

## The Biofilm Problem in Dairy Farms

In farm animals, bacteria able to form biofilm are involved with numerous persistent pathologies, often chronic and untreatable infections, such as arthritis in pigs or wound infections in equines (Gardner et al., 2011; Araújo et al., 2024). In milk-producing species, biofilm-forming bacteria are often associated with mastitis (Melchior, 2011; Pedersen et al., 2021; Araújo et al., 2024). Mastitis is caused by an IMI, which triggers an inflammatory response in the mammary gland (Halasa et al., 2007). Pathogenic microorganisms that cause IMI in dairy herds have been classified in terms of their mode of transmission (Blowey and Edmondson, 1995). Some originate in the environment (e.g., coliforms, *Strep. uberis*) and are associated mainly with clinical disease, whereas others are contagious (e.g., *Staph. aureus*, CNS, *Streptococcus agalactiae*) and persist as subclinical disease without visible signs (Bradley and Green, 2001; Zadoks et al., 2001; Pyörälä and Taponen, 2009). Although very visible and worrisome to dairy farmers, acute clinical forms of mastitis usually cause only short-term and relatively small drops in milk production (Gröhn et al., 2004). Subclinical mastitis occurs much more frequently, and the IMI often persists for long periods, sometimes the remaining life of the animal, causing inflammation and continued drop in milk production (Djabri et al., 2002).

Biofilm-forming ability is mentioned as a virulence factor, in the case of *Staph. aureus*, associated with persistence of the IMI but also with loss of sensitivity of the pathogen to antibiotics (Campos et al., 2022). Indeed, studies revealed that *Staph. aureus* isolates that express the gene coding the biofilm were able to better colonize the mammary gland, and thus persist (Cucarelла et al., 2001). Persistent CNS infections, which seem to occur at more advanced stages of lactation, are also associated with biofilm formation and loss of sensitivity to common disinfectants and antibiotics, requiring, for example, doses of penicillin G, novobiocin, or ceftiofur 4 to 2,048 times higher than the usual minimal inhibi-
tory concentration to eradicate the disease (Tremblay et al., 2013, 2014).

Several factors explain the loss of sensitivity of bacteria to antimicrobial agents in biofilms compared with the planktonic condition (Costerton et al., 1999; Hall-Stoodley and Stoodley, 2009). First, the polymer matrix acts as a physical size exclusion barrier and decreases or prevents diffusion of the compound into the biofilm, thus increasing the concentration of compound needed to destroy the cells. Second, the matrix bears surface electrostatic charges or hydrophobic groups that bind or block some antimicrobial agents and thereby enhance the barrier effect (Abdallah et al., 2014; Galié et al., 2018). Finally, and perhaps most importantly, because of the low concentrations of nutrients and oxygen inside a biofilm, cells therein are in a metabolic state close to dormancy (persister cells), making them resistant especially to antibiotics that work by inhibiting bacterial growth processes. Many cells may be weakened or even die, but as soon as the antibiotic is no longer present, the biofilm will be repopulated with viable cells, leading to chronicity of the infection (Humphreys and McBain, 2014). In addition, bacteria in mixed-species biofilms are collectively less sensitive to antibiotics because only one of the species present needs a β-lactamase for all the others to benefit from this resistance to β-lactam compounds (Hall-Stoodley and Stoodley, 2009).

The failure of some antibiotics to rid infected animals of pathogenic bacteria lodged in biofilm in vivo, as a result of resistance or enhanced tolerance, has stimulated efforts to develop new therapeutic compounds and strategies (Ceri et al., 2010). For example, the use of an antibiofilm molecule able to induce disruption of mature biofilm in combination with the initial antibiotic treatment could avoid the use of an increased concentration of antibiotics, and thus reduce the risk to disseminate antibiotic resistance (Ceri et al., 2010). Furthermore, the avenue of metagenomic and genomic analysis led to the discovery of new species within biofilm, enhancing our understanding of potential new bacterial targets and also giving insights on how these bacteria can develop resistances, which ultimately helps in developing new control strategies.

In addition, staphylococcal biofilms also tend to be less sensitive to iodine-based disinfectants, requiring contact times 2 to 10 times longer for eradication (Tremblay et al., 2014). In contrast, one compound found to be equally effective against several strains of CNS, whether planktonic or in biofilm, is chlorhexidine (Tremblay et al., 2014).

In addition to negatively affecting animal well-being and dairy yield, biofilm-forming bacteria are also able to persist on surface equipment in dairy farms and to induce a reduction of the microbiological quality of milk in dairy processing plants (Latorre et al., 2020).

## The Biofilm Problem in Dairy Processing Plants

Biofilm formation in dairy processing plants is a major concern, frequent source of microbial contamination of products, cause of economic losses, and potential menace to public health. Stainless steel (tanks, silos, pipelines, valves, plate heat exchangers, drains), polyethylene, polypropylene, rubber, and polytetrafluoroethylene (PTFE; floors, walls, gaskets, membranes) are all susceptible to colonization by microorganisms despite CIP procedures (Marchand et al., 2012; Yuan et al., 2019). Apart from insufficient or ineffective cleaning and sanitizing, several factors can influence the inherent ability of microbial cells to attach to these surfaces, such as material properties (presence of milk residues or conditioning film, roughness, topology, electrostatic charge, hydrophobicity), characteristics of the pumped fluid or food product (temperature, pH, oxygen level, salts and nutrients, microbial load, flow velocity, shear force), and microbial cell characteristics, such as hydrophobicity, motility, fimbriae, flagella and pili, lipopolysaccharides, exopolysaccharides, and the expression of signaling molecules (Simões et al., 2010; Phillips, 2016; Shemesh and Ostrov, 2020). In dairy processing environments, the presence of water and milk residues, the neutral pH, and the high nutrient content of milk, as well as the temperature and relative humidity ranges, are favorable conditions that can promote the formation of biofilm, and thus the persistence of bacteria (Fysun et al., 2019; Catania et al., 2023).

Biofilms found in dairy processing plants are generally multispecies biofilms and may consist of pathogens (*Cronobacter sakazakii*, *Enterobacter* spp., *Klebsiella* spp., *L. monocytogenes*, *Pseudomonas aeruginosa*, *Salmonella* spp., *Staph. aureus*, Shiga-toxin-producing *E. coli* O157:H7, *Streptococcus* spp.) and spoilage microorganisms (*Bacillus* spp., *Clostridium* spp., *Enterobacter* spp., *Pseudomonas* spp., *Streptococcus thermophilus*) found in raw milk (Oliveira et al., 2019; Panebianco et al., 2022). Rapid development of biofilms by these bacteria on ultrafiltration and reverse osmosis membranes and equipment surfaces may result in biofouling and increased shear stress, leading to release of cells into milk being processed (Kumar and Anand, 1998; Mogha et al., 2014; Chamberland et al., 2017). By causing repeated contaminations in this manner, they compromise the quality, the shelf life, and the safety of dairy products, and may lead to losses and ruinous recalls (Yuan et al., 2019). For instance, a persistent biofilm-forming isolate of *L. monocytogenes* reportedly survived 7 years in a Scandinavian dairy and contaminated cheese products (Unnerstad et al., 1996; Simões et al., 2010).

Whether the bacterial species is pathogenic or not, its persistence as biofilm in heat exchangers can reduce
heat transfer efficiency and result in milk not reaching the intended processing temperature, allowing pathogens and spoilage microorganisms to remain viable (Xu et al., 2021). Indeed, thermoduric bacteria of farm origin, such as *Bacillus licheniformis*, *Enterococcus faecalis*, and *Strep. thermophilus*, can resist milk processing and be found in processed dairy products through the formation of biofilms on heating equipment (Diarra et al., 2023). For example, strong biofilms formed by *Strep. thermophilus* in milk pasteurizers intended for cheese production have been reported as a source of contamination of processed dairy products and the cause of early quality defects observed in milk and cheese (fast acidification and texture modification; Bassi et al., 2017).

The persistence of mature multispecies biofilms can also accelerate the corrosion and deterioration of dairy processing line surfaces, making subsequent recolonization by microorganisms all the easier as well as increasing the frequency of equipment maintenance and replacement (Mogha et al., 2014; Gupta and Anand, 2018) and increasing reliance on disinfectants, thus promoting the development of microbial resistance to the active compounds and decreasing cleaning efficiency (Yuan et al., 2021). Mature multispecies biofilms (>168 h) have been also reported to be up to 10 to 1,000 times more resistant to biocides than planktonic cells and single-species biofilms, making them even more difficult to eradicate (Araújo et al., 2011; Phillips, 2016). By acting as a physical barrier, the extracellular polymeric substances of the biofilm matrix provide an effective protection against the penetration and the diffusion of antimicrobial agents to the targeted microbial cells located inside the biofilm (e.g., by size exclusion, degradation, electrostatic, or hydrophobic interactions; Abdallah et al., 2014; Galié et al., 2018). Biofilms can also be composed of microorganisms that are naturally resistant to biocides, have acquired resistance through plasmid transfer, or have reached a dormant or latent state, which further reduces their sensitivity to antimicrobial compounds and necessitates the development of new antibiofilm agents (Abdallah et al., 2014).

In the following section, various antibiofilm strategies currently used or studied in milk production and dairy processing are described.

# ANTIBIOFILMS

## Generalities

Considerable effort has been devoted to the development of new compounds or strategies for eradicating biofilms that are relevant to human health, which obviously includes more recently those encountered in the dairy chain. Antibiofilm strategies currently being studied

can be categorized as follows: (1) interfering with cell attachment or adhesins without focus on any specific bacterial species, for example, modifying material surface properties or using surfactants, with or without a biocide; (2) interfering with biofilm maturation, for example, blocking QS or other physiological signals using cyclic dinucleotides (di-cGMP) or small RNA; (3) disruptive modification of the bacterial cell membrane, for example, with antimicrobial peptides (e.g., bacteriocins) or of the extracellular matrix with hydrolytic enzymes, such as DNase I, dispersin B, or proteases (Beloin et al., 2014; Stewart, 2015; Verderosa et al., 2019; Asma et al., 2022; Kalia et al., 2023; Kilic and Bali, 2023; Kulshreshtha and Gupta, 2023; Hussaini et al., 2024). At least one approach focuses specifically on “persisters” (dormant cells) by targeting, for example, the stringent response mediator ppGpp (de la Fuente-Núñez et al., 2014).

Antibiofilm compounds that can be characterized as nontoxic, natural, and eco-responsible are attracting more and more attention (Kilic and Bali, 2023). These may be extracted from plants (e.g., essential oil), beehives or honey, marine products (e.g., chitosan), or microbial cultures, for example, bacteriocins and other peptides, enzymes, biosurfactants, or bacteriophages (Asma et al., 2022; Kilic and Bali, 2023; Hussaini et al., 2024). It may even be possible to repurpose some old drugs from the pharmaceutical industry (Stewart, 2015).

## Antibiofilms in Dairy Farms

Screening of molecules for efficacy as antibiofilm agents on dairy farms, especially for treatment of IMI, has recently exploded (Goetz et al., 2016). The various properties of these agents (type of compound, origin, mode, or spectrum of action) and the strategies proposed are summarized in Tables 1, 2, and 3.

The first strategy proposed to inhibit the development of a biofilm is to prevent its formation, by disrupting the attachment of planktonic bacteria to a surface, whether biotic (e.g., teat skin) or abiotic (e.g., milking equipment). This may involve targeting bacterial adhesion molecules (e.g., proteins, polysaccharides) or the physicochemical properties of the surface in question (Donlan, 2002). However, this strategy has not been adapted to IMI, and few studies of its application in milk production settings have been published (Aiemsaard et al., 2011; Xie et al., 2019). Whereas in mammary glands the desired goal is the eradication of already formed biofilm, in the case of milking equipment, milk storage or transport tanks, and processing surfaces in dairies, prevention is preferable to any cure (Teh et al., 2015). In addition to developing more strategies to avoid bacterial adhesion to surfaces, it would also be crucial to maintain good hygiene practices, improve them by using surfaces
<table><caption>**Table 1.** Inhibitors of attachment of planktonic bacteria to a biotic or abiotic surface studied in dairy farms (physicochemical modification of the surface prevents attachment and thus formation of biofilm)</caption><thead><tr><th>Antibiofilm</th><th>Form</th><th>Origin</th><th>Targeted microorganism</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Actinomycete culture<br>Lemongrass</td><td>Cell-free supernatant<br>Steam-distilled oil</td><td>Microbial<br>Plant</td><td><i>Staphylococcus epidermidis</i><br><i>Staphylococcus aureus</i></td><td>Inhibition of biofilm formation by interfering with cell adherence<br>Disrupts the bacterial cell surface, compromising initial attachment<br>to the substrate, and slowing biofilm formation</td><td>Xie et al., 2019<br>Atemsaard et al.,<br>2011</td></tr></tbody></table>

easier to clean, and regularly check the good condition of the latter (Marchand et al., 2012).

The second strategy proposed to inhibit biofilm formation is to disrupt biofilm architecture and thereby inhibit formation or induce dispersion (Table 2). Because cells thus returned to the planktonic condition usually regain their initial sensitivity to antibiotics, biofilm dispersers should be useful adjuncts to antibiotic-based attempts to cure IMI (Asli et al., 2017; Breser et al., 2018; Córdova-González et al., 2024). Other molecules appear suitable for preventing IMI, and thus avoiding the well-known problems associated with large-scale usage of antibiotics (Oliver and Murinda, 2012). These include proteinase K, which cleaves surface-bound proteins, and thus disrupts the architecture of *Staph. aureus* biofilm (Shukla and Rao, 2017). However, numerous natural compounds of microbial origin, such as bacteriocins and other peptides (Duarte et al., 2018; Pérez-Ibarreche et al., 2021; Zhang et al., 2021; Yang et al., 2022), or of plant origin, such as essential oils (Budri et al., 2015; Cerioli et al., 2018; Abd El-Aziz et al., 2021; Abd El-Hamid et al., 2023), have received more attention from researchers (Table 2).

Finally, interruption of biological signals used by bacteria (mechanical, environmental, intracellular, or cell-density-based) appears to offer both prophylactic and therapeutic means of suppressing biofilm (Table 3). So far, the most promising example of this approach appears to be the development of inhibitors of QS (González-Ortiz et al., 2014; Qiu et al., 2022; Liu et al., 2023), which may be small molecules, nonpeptide, peptide, protein, plant fibers, or whole bacterial cells, all either inhibiting or modifying signal generation or blocking signal reception and hence the cellular interactions necessary for biofilm formation.

In most cases, more study is needed to evaluate their in vivo efficacy for the treatment of IMI in dairy cows as well as to identify the active molecules, determine their toxicity, and establish the optimal formulation and mode of administration. For example, antibiofilm-based formulations, such as ointments, intramammary suspensions, or dipping solutions, could be developed. Depending on the galenic form used, a treatment regimen will be decided. For intramammary suspensions, the product could be administered using intramuscular or intravenous injections and used during the dry period, to prevent the onset of new IMI or to treat preexisting IMI. For the dipping solution or ointments, the molecule could be administered topically after milking to dislodge bacteria from the teat apex and thus prevent the onset of IMI. Finally, the efficacy of the product should be evaluated by measuring pharmacokinetics (bioavailability, maximal concentration, distribution volume, half-life, and clearance) and pharmacodynamics (therapeutic window) parameters to receive approval from regulatory authorities.
<table><caption>**Table 2.** Biofilm architecture disruptors studied in dairy farms¹</caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Solanum lasiocarpum root extract with synthesized silver/silver chloride nanoparticles</td><td>Nanomaterial</td><td>Biological engineering</td><td>Staphylococcus haemolyticus</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Wintachai et al., 2024</td></tr><tr><td>Graphene oxide</td><td>Nanomaterial</td><td>Biological engineering</td><td>Staphylococcus aureus</td><td>Inhibition of biofilm formation</td><td>Saeed et al., 2023</td></tr><tr><td>Syzygium aromaticum, Cinnamomum verum, Emblica officinalis, Terminalia belerica, Terminalia chebula, and Cymbopogon citratus</td><td>Polyherbal nanocolloids</td><td>Biological engineering</td><td>Acinetobacter junii, Klebsiella pneumoniae, Pseudomonas stutzeri, and Acinetobacter baumannii</td><td>Inhibition of biofilm formation</td><td>Ranjani et al., 2022</td></tr><tr><td>Silver and copper nanoparticles</td><td>Nanomaterial</td><td>Biological engineering</td><td>Mastitis pathogens</td><td>Inhibition of biofilm formation</td><td>Lange et al., 2021</td></tr><tr><td>Bioengineered nisin derivative</td><td>Antimicrobial peptide</td><td>Biological engineering</td><td>Streptococcus uberis</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Pérez-Ibarreche et al., 2021</td></tr><tr><td>Chitosan nanoparticles</td><td>Nanomaterial</td><td>Biological engineering</td><td>Staph. aureus</td><td>Inhibition of biofilm formation</td><td>Orellano et al., 2021</td></tr><tr><td>Zinc-aluminum-layered double hydroxide intercalated with gallic acid</td><td>Nanomaterial</td><td>Biological engineering</td><td>Escherichia coli, K. pneumoniae, Staph. aureus, coagulase-negative staphylococci, Listeria monocytogenes</td><td>Inhibition of biofilm formation</td><td>Mohammed et al., 2020</td></tr><tr><td>Chitosan nanoparticles</td><td>Nanomaterial</td><td>Biological engineering</td><td>Streptococcus dysgalactiae</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Rivera Aguayo et al., 2020</td></tr><tr><td>BrpA-like proteins</td><td>Protein</td><td>Biological engineering</td><td>E. coli</td><td>Inhibition of biofilm formation by decreasing transcription of biofilm-associated genes bcsA, csgA, fliC, fimA, motA, and wcaF</td><td>Alves-Barroco et al., 2019</td></tr><tr><td>Silver-nanoparticle-decorated quercetin nanoparticles</td><td>Nanomaterial</td><td>Biological engineering</td><td>Staphylococcus xylosus</td><td>Inhibition of biofilm formation by targeting imidazole glycerophosphate dehydratase</td><td>Yu et al., 2018</td></tr><tr><td>Baicalin</td><td>Protein</td><td>Biological engineering</td><td>Staph. aureus</td><td>Disruption of mature biofilm by targeting extracellular polysaccharide matrix</td><td>Chen et al., 2017</td></tr><tr><td>Biofilm clippers</td><td>Enzyme</td><td>Chemical synthesis</td><td>Staph. aureus</td><td>Disruption of mature biofilm</td><td>Thiruvengadam et al., 2019</td></tr><tr><td>Polyhexamethylene biguanide</td><td>Antimicrobial polymer</td><td>Chemical synthesis</td><td>Staph. aureus</td><td>Inhibition of biofilm formation and disruption of mature biofilm by cleavage of surface proteins</td><td>Kamaruzzaman et al., 2017</td></tr><tr><td>Proteinase K</td><td>Enzyme</td><td>Chemical synthesis</td><td>Staph. aureus</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Shukla and Rao, 2017</td></tr><tr><td>Enrofloxacin hydrochloride-dihydrate/ dimethyl sulfoxide/chitosan</td><td>Cationic polymer + fluoroquinolone</td><td>Chemical synthesis, animal</td><td>Staph. aureus</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Córdova-González et al., 2024</td></tr><tr><td>Linalool, carvacrol, and eugenol</td><td>Essential oils</td><td>Chemical synthesis, plant</td><td>Staph. aureus</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Abd El-Hamid et al., 2023</td></tr><tr><td>Low-molecular-weight chitosan</td><td>Polyoside</td><td>Animal</td><td>Staphylococcus spp.</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Felipe et al., 2019</td></tr><tr><td>Chitosan and cloxacillin</td><td>Cationic polymer, with β-lactam</td><td>Animal</td><td>CNS</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Breser et al., 2018</td></tr><tr><td>Chitosan, alone or with antibiotic</td><td>Animal</td><td>Boitrops jararacussu</td><td>Staph. aureus</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Asli et al., 2017</td></tr><tr><td>C-type lectin from pit viper venom</td><td>Protein</td><td></td><td>Staph. aureus, Staphylococcus epidermidis, Staphylococcus chromogenes, Staphylococcus hyicus, Streptococcus agalactiae, E. coli</td><td>Inhibition of biofilm formation</td><td>Klein et al., 2015</td></tr></tbody></table>
<table><caption>**Table 2 (Continued).** Biofilm architecture disruptors studied in dairy farms¹</caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Exopolysaccharides produced by <i>Bacillus</i> spp.<br>NZ2114</td><td>Carbohydrate<br>Antimicrobial peptide</td><td>Microbial<br>Microbial</td><td><i>Staph. aureus</i><br><i>Strep. dysgalactiae</i></td><td>Inhibition of biofilm formation<br>Inhibition of biofilm formation, disruption of early and mature biofilm, elimination of encapsulated "persister" bacteria<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation, disruption of early and mature biofilm, elimination of encapsulated "persister" bacteria<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation</td><td>Sabino et al., 2023<br>Yang et al., 2022<br>Song et al., 2021<br>Zhang et al., 2021</td></tr><tr><td>B_SauM_SDQ<br>Recombinant fungal defensinlike peptide-P2</td><td>Bacteriophage<br>Antimicrobial peptide</td><td>Microbial<br>Microbial</td><td><i>Staph. aureus</i><br><i>Strep. dysgalactiae</i></td><td>Inhibition of biofilm formation, disruption of early and mature biofilm, elimination of encapsulated "persister" bacteria<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation, disruption of early and mature biofilm, elimination of encapsulated "persister" bacteria<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation</td><td>Shan et al., 2020<br>Santos et al., 2018<br>Duarte et al., 2018</td></tr><tr><td>Staphylococcal bacteriophage lysin<br>CHAPk<br><i>Trichoderma asperelloides</i><br>Hyicin 4244</td><td>Peptidoglycan hydrolase<br>Ethanolic extract<br>Antimicrobial peptide</td><td>Microbial<br>Microbial<br>Microbial</td><td><i>Strep. agalactiae</i><br><i>Staph. aureus</i><br><i>Staphylococcus</i> spp.<br><i>Trueperella pyogenes</i></td><td>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation</td><td>da Silva Duarte et al., 2018<br>Isaac et al., 2017</td></tr><tr><td>T4 virus<br>Commensal coagulase-negative <i>Staphylococcus</i><br><i>Staph. chromogenes</i>, and <i>Staphylococcus simulans</i></td><td>Cell-free supernatant<br>Bacteria</td><td>Microbial<br>Microbial</td><td><i>Staph. aureus</i> and CNS<br><i>Staphylococcus</i> spp.<br><i>Staph. aureus</i></td><td>Inhibition of biofilm formation<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Immunization by vaccine inducing high antibody production against poly N-acetyl-D-glucosamine (PNAG)<br>Inhibition of biofilm formation</td><td>Goetz et al., 2017<br>Pérez et al., 2009</td></tr><tr><td>Biofilm-producing <i>Staph. aureus</i> strain</td><td>Inactivated cells</td><td>Microbial</td><td><i>Staph. aureus</i></td><td>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation</td><td>Sungkatavat et al., 2023<br>Guzmán-Rodríguez et al., 2024<br>Mao et al., 2023</td></tr><tr><td>Piper betle <i>Agave fructans</i></td><td>Extract<br>Prebiotic</td><td>Plant<br>Plant</td><td><i>Staphylococcus</i> spp.<br><i>Staph. aureus</i></td><td>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation</td><td>Chuprom et al., 2023<br>Xu et al., 2022</td></tr><tr><td>Baicalein<br><i>Knema retusa</i></td><td>Flavonoid compound<br>Wood extract</td><td>Plant<br>Plant</td><td><i>Staph. aureus</i><br><i>Staph. aureus and Staph. haemolyticus</i></td><td>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation and disruption of mature biofilm<br>Inhibition of biofilm formation by downregulation of pili biosynthesis gene expression needed for biofilm development<br>Inhibition of biofilm formation</td><td>Purgato et al., 2021<br>Abd El-Aziz et al., 2021</td></tr><tr><td>Caffeic acid<br><i>Salvinia auriculata</i><br>Cinnamon oil</td><td>Phenolic compound<br>Dried weed<br>Essential oil</td><td>Plant<br>Aquatic fern<br>Plant</td><td><i>Staph. aureus</i><br><i>Strep. agalactiae</i></td><td>Inhibition of biofilm formation by downregulating the transcription of the biofilm-associated genes including <i>svrA</i>, <i>fsbC</i>, <i>neuA</i>, and <i>cpsE</i><br>Inhibition of biofilm formation<br>Inhibition of biofilm formation</td><td>Ren et al., 2020<br>Shang et al., 2020</td></tr><tr><td>Oxytropis glabra DC.</td><td>Decoction</td><td>Plant</td><td><i>Staph. epidermidis</i></td><td>Inhibition of biofilm formation by downregulating the transcription of the biofilm-associated genes including <i>svrA</i>, <i>fsbC</i>, <i>neuA</i>, and <i>cpsE</i><br>Inhibition of biofilm formation<br>Inhibition of biofilm formation</td><td>Manzur et al., 2020<br>Wang et al., 2024<br>Gomes et al., 2019</td></tr><tr><td>Tea saponin<br><i>Mangifera indica</i> L.<br>1-Hydroxyanthraquinone<br>Hydromethanolic extracts</td><td>Extract<br>Anthraxinone<br>Extract</td><td>Plant<br>Plant<br>Plant</td><td><i>Staphylococcus</i> spp.<br><i>Staph. xylosus</i><br><i>Staph. aureus</i></td><td>Inhibition of biofilm formation by downregulating the transcription of the biofilm-associated genes including <i>svrA</i>, <i>fsbC</i>, <i>neuA</i>, and <i>cpsE</i><br>Inhibition of biofilm formation<br>Inhibition of biofilm formation</td><td>Continued</td></tr></tbody></table>
<table><caption>**Table 2 (Continued).** Biofilm architecture disruptors studied in dairy farms<fnref n="1" /></caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Isoliquiritigenin</td><td>Flavonoid</td><td>Plant</td><td>Staph. xylosus</td><td>Inhibition of biofilm formation by downregulating <i>hisB</i> gene expression</td><td>Qu et al., 2019</td></tr><tr><td>Stigmata maydis</td><td>Extract</td><td>Corn silk</td><td>Staph. aureus</td><td>Inhibition of biofilm formation by regulating <i>icaR</i> gene expression</td><td>Shang et al., 2019</td></tr><tr><td>Calliandra surinamensis leaf pinnulae lectin</td><td>Protein</td><td>Plant</td><td>Staphylococcus spp.</td><td>Inhibition of biofilm formation</td><td>Procópio et al., 2019</td></tr><tr><td>Minthostachys verticillata (Griseb.) Epling</td><td>Essential oil limonene</td><td>Plant</td><td>E. coli, Bacillus pumilus, Enterococcus faecium</td><td>Inhibition of biofilm formation and disruption of mature biofilm</td><td>Cerioli et al., 2018</td></tr><tr><td>Plectranthus ornatus Codd. Sophora alopecuroides</td><td>Extract Alkaloids</td><td>Plant</td><td>Staph. aureus</td><td>Inhibition of biofilm formation</td><td>Nascimento et al., 2017</td></tr><tr><td>Citral, linalool, decanal, and valencene</td><td>Orange oil</td><td>Plant</td><td>Staph. epidermidis</td><td>Inhibition of biofilm formation</td><td>Li et al., 2016</td></tr><tr><td>Citrus-derived oil</td><td>Orange oil</td><td>Plant</td><td>Staph. aureus</td><td>Disruption of mature biofilm</td><td>Federman et al., 2016b</td></tr><tr><td>Syzygium aromaticum and Cinnamomum zeylanicum</td><td>Essential oils and major components</td><td>Plant</td><td>Staph. aureus</td><td>Inhibition of biofilm formation</td><td>Federman et al., 2016a</td></tr><tr><td>Sanguisorba officinalis L.</td><td>Ethanol extract</td><td>Plant</td><td>Staph. aureus</td><td>Inhibition of biofilm formation by increasing <i>icaR</i> transcripts to inhibit the <i>icaI</i>DBC transcription operon, which encodes the polysaccharide intercellular adhesin synthetases</td><td>Budri et al., 2015</td></tr></tbody></table>

<table><caption>**Table 3.** Use of disruptors of intercellular and intracellular signals as antibiofilm agents in dairy farms (signal inhibitors or modifiers and signal reception blockers)</caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Azithromycin</td><td>Macrolide antibiotic</td><td>Chemical synthesis</td><td>Staphylococcus xylosus</td><td>Inhibition of biofilm formation by downregulating proteins involved in the histidine biosynthesis</td><td>Ding et al., 2018</td></tr><tr><td>Cefquinome</td><td>Fourth-generation cephalosporin</td><td>Chemical synthesis</td><td>Staph. xylosus</td><td>Inhibition of biofilm formation by downregulating an enzyme involved in histidine metabolism</td><td>Zhou et al., 2018</td></tr><tr><td>di-GMP</td><td>Secondary messenger</td><td>Chemical synthesis</td><td>Staphylococcus aureus</td><td>Inhibition of intercellular adhesion</td><td>Brouillette et al., 2005; Karaolis et al., 2005</td></tr><tr><td>Calcium</td><td>Secondary messenger</td><td>Chemical synthesis</td><td>Staph. aureus</td><td>Inhibition of BAP-dependent multicellular behavior</td><td>Arrizubeta et al., 2004</td></tr><tr><td>Commensal Bacillus subtilis</td><td>Cell-free supernatant</td><td>Microbial</td><td>Staph. aureus</td><td>Inhibition of biofilm formation by regulating QS signaling</td><td>Qiu et al., 2022</td></tr><tr><td>Wheat bran</td><td>Extract</td><td>Plant</td><td>Staph. aureus</td><td>Inhibition of biofilm formation by interfering with bacterial QS systems</td><td>González-Ortiz et al., 2014</td></tr><tr><td>Luteolin</td><td>Flavonoid</td><td>Plant</td><td>Staph. aureus</td><td>Inhibition of agr QS or hemolysin</td><td>Liu et al., 2023</td></tr></tbody></table>

<footnote n="1">¹This category of antibiofilm molecules acts by blocking biofilm maturation or by causing premature sloughing of bacterial cells.</footnote>
Antibiofilms in Dairy Processing Plants

Cleaning of dairy processing equipment surfaces is cur-
rently ensured by automated circulation of strong alkaline
solution (e.g., sodium hydroxide, potassium hydroxide,
sodium carbonate) and acids (e.g., nitric, phosphoric,
hydrochloric, citric, sulfuric) at high temperature and
high fluid velocity alternating with rinsing cycles. This
is known as CIP. Though not mandatory, a subsequent
sanitation step is often carried out using chemicals (e.g.,
aldehyde, chlorine, iodine-based compounds, peracetic
acid, hydrogen peroxide, ozone, quaternary ammonium
compounds) at predetermined concentrations and contact
times (Mogha et al., 2014; Pant et al., 2023). The CIP
process eliminates organic matter residues and usually
prevents microbial attachment and biofilm formation in
tanks, heat exchangers, and pipes (Thomas and Sathian,
2014; Teh et al., 2015; Pant et al., 2023). The effective-
ness of the chemical substances used in these procedures
depends on several factors, such as the mechanism of
action of the chemical compound and its concentration,
contact time, temperature, pH, and water hardness, as
well as the presence or absence of organic and inor-
ganic residues on the surface, maturity of the biofilm,
and microbial species constituting the biofilms (Srey et
al., 2013). In the food and dairy sectors, a sanitizer or
disinfectant must also be chemically stable with a broad
spectrum, noncorrosive to surfaces, easy to use, and af-
fordable (Watkinson, 2009; Gaulin et al., 2011; Thomas
and Sathian, 2014). Among the chemicals mentioned pre-
viously, chlorine-based oxidizing sanitizers (50–500 mg/
kg) and peracetic acid-based sanitizers (50–350 mg/kg)
are the most used in dairy processing plants, and several
studies have shown their effectiveness against planktonic
cells and biofilms, as well as their ineffectiveness (Table
4) in certain cases (Gaulin et al., 2011; Megahed et al.,
2019). Through dissociation into hypochlorous acid
(HClO) and hypochlorite ions (ClO⁻), sodium hypochlo-
rite oxidizes sulfhydryl groups and thereby denatures
enzymes, proteins, peptides, amino acids, lipids, and
DNA, thus irreversibly damaging essential components
of microbial cells and biofilm matrices, but it also reacts
with food constituents, including milk residues present
on equipment surfaces, thereby losing antimicrobial
potency and sometimes producing harmful compounds,
such as chloramines and trihalomethanes (Lineback
et al., 2018; Silva et al., 2018; Carrascosa et al., 2021;
Zhang et al., 2024). Peracetic acid is recognized as an
eco-friendly alternative to sodium hypochlorite, because
it similarly attacks microbial cells and the biofilm ma-
trix by oxidizing thiol and amine groups in addition to
creating free hydroxyl and peroxyl radicals, but without
generating toxic byproducts from organic residues (Wat-
kinson, 2009; Kerkaert et al., 2011; Chino et al., 2017;

Megahed et al., 2019). However, because standard con-
centrations of biocide are often sublethal and the mode of
action is generally nonspecific (multitargeted), penetra-
tion of the polymer matrix is incomplete and biofilm is
not eradicated, allowing the bacteria therein to develop
resistance and cross-resistance to the biocides (Bridier et
al., 2011; György et al., 2023). Furthermore, it has been
observed that dead microbial cells from biofilms treated
with organic peroxyacids can remain attached to polysty-
rene surfaces and form a conditioning film that facilitates
recolonization, further underscoring the importance of
completely eliminating biofilm matrix and cells from
equipment surfaces to prevent repeated contamination of
dairy products (Goetz et al., 2022a).

Ozone, organic peroxyacids, bacteriophages and
proteins therefrom, bacteriocins, hydrolytic enzymes,
quorum-sensing inhibitors, essential oils, and steel coat-
ings (Tables 4 and 5) are relatively recent among the
strategies being investigated to overcome biofilm persis-
tence (Galié et al., 2018; Gutiérrez et al., 2019). Ozone,
aqueous and gaseous, despite a mode of action similar to
sodium hypochlorite and peracetic acid, reportedly inac-
tivates dairy-related biofilms formed on stainless steel,
polypropylene, polystyrene, and glass by *Bacillus* spp.,
*L. monocytogenes*, *Pseudomonas* spp., *Salmonella* spp.,
and *Staph. aureus* (Panebianco et al., 2022). Highly spe-
cific phages appear to disrupt biofilms formed by *Staph.
aureus* (phages K, ISP), *L. monocytogenes* (phage P100),
and *E. coli* O157:H7 (phage BPECO 19), and depolymer-
ases, endolysins, and virion-associated peptidoglycan
hydrolases can hydrolyze bacterial exopolysaccharides,
inhibit biofilm formation, and damage dormant and per-
sistent cells (Bridier et al., 2011; Gutiérrez et al., 2019).

Some combinations of novel promising strategies for
dairy biofilm control have also been explored. For exam-
ple, Radwan et al. (2022) have reported the synergistic
effect of proteinase K coupled with biosynthesized zinc
oxide nanoparticles, shown to generate reactive oxygen
species (ROS), inhibit respiratory enzymes, and damage
cell membranes, in disrupting biofilms formed by the
dairy sporulating bacteria *Bacillus* spp. and *Brevibacil-
lus* spp. on stainless steel coupons. A growing number of
studies have described the similar antimicrobial effects
of zinc compounds and nanoparticles (e.g., Zn²⁺, ZnCl₂,
ZnO, ZnS) on biofilms formed by pathogenic bacteria
(e.g., *P. aeruginosa*, *Staph. aureus*, *E. coli*, *Salmonella*
Typhimurium), as well as the increased sensitivity to heat
of *Bacillus cereus* and *Bacillus subtilis* after exposure to
zinc (Wu et al., 2013; Hutchings et al., 2020; Husain et
al., 2022; Bianchini Fulindi et al., 2023).

Modification of material surface properties—for
example, reducing roughness or hydrophobicity using
antimicrobial coatings or antiadhesive compounds (e.g.,
biosurfactants, peptides)—is another possible approach
<table><caption>**Table 4.** Biofilm architecture disruptors studied in dairy processing plants<fnref n="1" /></caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Proteinase K coupled with biosynthesized zinc oxide nanoparticles</td><td>Enzyme and nanoparticles</td><td>Biological synthesis and engineering</td><td><i>Bacillus</i> spp. and <i>Brevibacillus</i> spp.</td><td>Eradication of biofilm on stainless steel by a synergistic mechanism<br>Proteinase K: Dispersion by proteolysis<br>ZnO: Generation of reactive oxygen species, inhibition respiratory enzymes, cell membranes damage<br>Ineffective at the concentrations tested in minimum biofilm eradication concentration (MBEC) plate assay (Biofilm Inoculator microplate, InnovoTech)<br>Disruption of matrix components and complete eradication of biofilm formed on stainless steel</td><td>Radwan et al., 2022</td></tr><tr><td>Peracetic acid and sodium hypochlorite</td><td>Organic peroxyacid and chlorine</td><td>Chemical synthesis</td><td><i>Acinetobacter baumannii</i>, <i>Pseudomonas</i> spp.</td><td></td><td>Sanschagrin et al., 2024</td></tr><tr><td>Enzymatic treatment (DNase I or lipase) before CIP (NaOH and nitric acid solutions)</td><td>Enzymes</td><td>Chemical synthesis</td><td><i>Brevibacillus agri</i></td><td></td><td>Zhang et al., 2024</td></tr><tr><td>Pertlactic acid, perpropionic acid, peracetic acid, commercial peracetic acid-based disinfectant, hydrogen peroxide Gaseous ozone</td><td>Organic peroxyacids</td><td>Chemical synthesis</td><td><i>Pseudomonas azotoformans</i>, <i>Serratia liquefaciens</i>, <i>Brevundimonas vesicularis</i>, <i>Listeria monocytogenes</i></td><td>Biofilm disruption at recommended or tested concentrations in MBEC assay</td><td>Goetz et al., 2022a</td></tr><tr><td>Inorganic oxidizing agent</td><td></td><td>Chemical synthesis</td><td><i>L. monocytogenes</i></td><td>Effective at high concentration (50 mg/kg) on planktonic cells, biofilm prevention, and removal partial (polystyrene microtiter plate)</td><td>Panebianco et al., 2021</td></tr><tr><td>Zinc compound</td><td>Organic peroxyacid</td><td>Chemical synthesis</td><td><i>Bacillus cereus</i> and <i>Bacillus subtilis</i></td><td>Inhibition of formation, disruption of bundles by downregulation of extracellular matrix component synthesis</td><td>Hutchings et al., 2020</td></tr><tr><td>Peracetic acid</td><td>Organic peroxyacid</td><td>Chemical synthesis</td><td><i>Staphylococcus aureus</i> and <i>L. monocytogenes</i></td><td>Inactivation of biofilm on stainless steel</td><td>Lee et al., 2016</td></tr><tr><td>Benzalkonium chloride, sodium hypochlorite, and iodophor Citric acid</td><td>Organic quaternary ammonium, chlorine, iodine complex</td><td>Chemical synthesis</td><td><i>Pseudomonas aeruginosa</i></td><td>Biocides were ineffective at recommended concentrations against biofilms preformed in polystyrene microtiter plates</td><td>Pagedar and Singh, 2015</td></tr><tr><td>Aerosolized peracetic acid and sodium hypochlorite</td><td>Organic peroxyacid and alkaline inorganic chemical</td><td>Chemical synthesis</td><td><i>Staph. aureus</i></td><td>Prevention of biofilm formation, removal (on stainless steel and polystyrene)</td><td>Akbas and Kokumer, 2015</td></tr><tr><td>Peracetic acid and reuterin</td><td>Organic peroxyacid and bacteriocin</td><td>Chemical synthesis and microbial</td><td><i>Escherichia coli</i> O157:H7, <i>Salmonella Typhimurium</i>, <i>L. monocytogenes</i></td><td>Inactivation of mature biofilm on stainless steel and polyvinyl chloride (PVC) coupons</td><td>Park et al., 2012</td></tr><tr><td>BM173 from <i>Lactobacillus crustorum</i> MN047</td><td>Bacteriocin</td><td>Microbial</td><td><i>P. azotoformans</i>, <i>Serr. liquefaciens</i>, and <i>Bacillus licheniformis</i></td><td>Biofilm disruption at recommended or tested concentrations in MBEC assay</td><td>Niboucha et al., 2024</td></tr><tr><td>Rhamnolipids</td><td>Biosurfactant</td><td>Microbial</td><td><i>E. coli</i> O157:H7</td><td>Inhibition of biofilm formation by forcing pores in cell membrane, cell lysis (in polystyrene microtiter plates)</td><td>Qiao et al., 2021</td></tr><tr><td>Phage BPECO 19</td><td>Lytic bacteriophage</td><td>Microbial</td><td><i>E. coli</i> O157:H7</td><td>Disruption and dispersion (in polystyrene microtiter plates) by reducing surface or interfacial tensions</td><td>Silva et al., 2017</td></tr><tr><td>Phage K and modified derivatives</td><td>Essential oil</td><td>Microbial</td><td><i>Staph. aureus</i></td><td>Inactivation of cells in biofilm formed on various food contact surfaces</td><td>Sadekuzzaman et al., 2017</td></tr><tr><td><i>Litsea cubeba</i></td><td></td><td>Natural</td><td><i>Cronobacter sakazakii</i></td><td>Prevention of biofilm, disruption by lysis (in polystyrene microtiter plates)</td><td>Kelly et al., 2012</td></tr><tr><td></td><td></td><td></td><td></td><td>Disruption of biofilm on stainless steel coupons (by intracellular ROS production and cell damage due to membrane permeabilization)</td><td>Wang et al., 2022</td></tr></tbody></table>

<footnote n="1">¹This category of antibiofilm molecules acts by blocking biofilm maturation or by causing premature dispersion of bacterial cells.</footnote>
<table><caption>**Table 5.** Inhibitors of planktonic bacterial attachment to abiotic surfaces studied in dairy processing plants (surface modification prevents attachment and thus formation of biofilm)</caption><thead><tr><th>Antibiofilm</th><th>Type</th><th>Origin</th><th>Targeted microorganism(s)</th><th>Mechanism of action</th><th>Reference</th></tr></thead><tbody><tr><td>Peptide coating</td><td>Peptide</td><td>Chemical synthesis</td><td><i>Bacillus licheniformis</i>, <i>Pseudomonas aeruginosa</i></td><td>Prevention of cell attachment, biofilm formation, and maturation by modification of the surface properties of stainless steel</td><td>Friedlander et al., 2019</td></tr><tr><td>Surfactin and rhamnolipids</td><td>Biosurfactant</td><td>Microbial</td><td><i>Staphylococcus aureus</i>, <i>Listeria monocytogenes</i>, and <i>Salmonella enterica</i></td><td>Inhibition of cell adhesion by lowering surface hydrophobicity, disruption of biofilm formed (in polystyrene microtiter plates)</td><td>Zezzi do Valle Gomes and Nitschke, 2012</td></tr></tbody></table>

to prevent the attachment of microbial cells to surfaces and subsequent formation of biofilms (Tables 4 and 5; Zezzi do Valle Gomes and Nitschke, 2012; Silva et al., 2017; Friedlander et al., 2019; Shemesh and Ostrov, 2020). However, this has yet to be investigated in situ, and studies are necessary to verify their applicability and efficacy in dairy processing plants. Indeed, several strict governmental and safety regulations could limit the potential for application of these new strategies. As an example, a new antibiofilm molecule should be studied and tested in depth to determine its effective microbial load reduction capacity, as well as its toxicity and compatibility properties, and this within a threshold of pre-established maximum concentrations that can be in contact with food surfaces, before being approved by governmental authorities and used in dairy industries.

## PERSPECTIVES

An abundant literature focuses on problematic bacteria in human and veterinary medicine and their ability to form persistent biofilms. The various components and developmental stages of bacterial biofilms have been also described in detail. This knowledge has allowed the development of a variety of antibiofilm compounds that target specifically the stages of attachment, maturation, and dispersion, as well as taking into consideration biofilm composition, which may vary considerably from one species to another. The discovery of promising new antibiofilm molecules is expected to accelerate over the next years thanks to the use of powerful new technologies, such as high-throughput screening, nanomaterials, artificial intelligence, and CRISPR. Because antibiofilm molecules differ from antibiotics and disinfectants in terms of mechanism and physiological target, their use in the dairy industry is proposed with the expectation of obtaining synergy and thus allowing the latter agents to be effective at lower concentrations. This needs to be borne out in studies of the efficacy of the new compounds in situ as well as the limitations (e.g., cost, specificity, biological risk) associated with their use on cow IMI or in dairy processing plants. In addition to the development of new antibiofilm strategies, further studies should investigate the microbial community and their physiological state within biofilms in the dairy industry. Indeed, biocides are often tested against pathogenic bacteria, such as *Staph. aureus* or *P. aeruginosa*. Furthermore, it would also be important to evaluate whether these new molecules could induce a viable but nonculturable (VBNC) state in some bacterial species. Indeed, studies have highlighted that disinfection methods using acetic acid were able to induce VBNC cells, allowing them to persist in the environment (Xiao et al., 2025). Finally, the environmental conditions on the farm or in
dairy processing plants are also important to consider, as a recent study highlighted that high temperatures and pH values increase biofilm formation (Roy et al., 2021). In addition, hygiene measures at the farm should be maintained and improved, using, for example, surfaces that are easier to clean up to avoid the adhesion of bacteria and, thus, biofilm-related infections.

## CONCLUSIONS

The investigation of natural approaches to prevent mastitis and to ensure the safety and quality of dairy products constitutes a sustainable approach likely to meet societal expectations and contribute to the development of food qualities from the upstream stage by proposing strategies that are more respectful of the environment (preservation of microbial biodiversity, reduction of chemical products and antimicrobials released in the environment), animal health and welfare (prevention of infections), and human health (food safety, contribution to the fight against antimicrobial resistance through the exploration of alternative approaches).

## NOTES

Coralie Goetz is the recipient of funding from the European Union Horizon 2020 research and innovation program (Brussels, Belgium) under the Marie Skłodowska-Curie grant agreement no. 899546. The authors thank Novalait (Quebec, Canada), the FRQNT Op+Lait Strategic Cluster (St-Hyacinthe, Canada), and the Galactinnov International Research Network (St-Hyacinthe), which made this collaboration possible. Finally, we thank Stephen Davids (SENSICULTURE FY Inc., Québec, QC, Canada) for proofreading the manuscript and translating portions thereof. Because no animals were used, approval by an Institutional Animal Care and Use Committee was not required. The authors have not stated any conflicts of interest.

**Nonstandard abbreviations used:** CIP = clean-in-place; QS = quorum sensing; ROS = reactive oxygen species; VBNC = viable but nonculturable.

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ORCIDS

Coralie Goetz, https://orcid.org/0000-0001-5073-1141

Laurie Sanschagrin, https://orcid.org/0009-0002-6457-9069

Eric Jubinville, https://orcid.org/0000-0001-5010-5087

Mario Jacques, https://orcid.org/0000-0001-6294-0848

Julie Jean, https://orcid.org/0000-0003-2553-4204