**4. Fighting against biofilms with nonconventional methods**

Since biofilms act as a barrier that protects the embedded cells against cleaning and disinfecting agents [51], the control of biofilm is an issue that is currently

### *Bacterial Biofilms*

addressed to find effective solutions that can prevent biofilm formation or eliminate the already formed one. Biocontrol of biofilms by using bacteriocins, disruptive enzymes, essential oils, or bacteriophages is gaining importance, as well as using nanoemulsions and nanoparticles. These new methods are promising strategies with remarkable results in the fight against biofilms.

## **4.1 Bacteriocins used to control biofilms**

Bacteriocins are antimicrobial peptides ribosomally produced by an extensive range of bacteria to inhibit or kill competing microorganisms in a micro-ecological system [52, 53]. The most studied bacteriocin and the only one allowed presently as food-grade additive is nisin, a lantibiotic with proven effects against many Grampositive bacteria including foodborne pathogens [54]. This bacteriocin was shown to penetrate the biofilm formed by *S. aureus* and permeate the sessile bacterial cells by real-time monitoring [55]. Moreover, nisin and its bioengineered derivatives were able to enhance the capability of conventional antibiotics such as chloramphenicol of decreasing *S. aureus* biofilm viability [56]. Nevertheless, a study assessing the effect of neutral electrolyzed water and nisin and their combination against listerial biofilm on glass and stainless steel surfaces indicated the potency of this bacteriocin to improve the efficacy of sanitizers used in food industry [57]. Nisin was also indicated to be effective against biofilms formed by Gram-negative bacteria such as *Salmonella* typhimurium when combined with P22 phage and EDTA, a synergistic combination that reduced 70% of the mature biofilm [58].

Another way to prevent biofilms development is represented by the adsorption of these bioactive compounds on the surfaces that come into contact with foods [59]. In this case, Nisaplin adsorbed to three types of food-contact surfaces commonly encountered in food processing plants, namely stainless steel, polyethylene terephthalate (PET), and rubber, reduced the adhesion ability of food-isolated *L. monocytogenes* strains [60]. Other studies showing the efficacy of nisin in preventing surface colonization by *L. monocytogenes* were conducted by Daeschel et al. [61] and Bower et al. [62].

A bacteriocin found to markedly inhibit the biofilm formed by *S. aureus* is sonorensis, a member of the heterocycloanthracin subfamily produced by *Bacillus sonorensis* MT93 [63].

### **4.2 Disruptive enzymes for fighting against biofilms**

Disruptive enzymes, such as proteases, glycosidases, amylases, cellulases, or DNAses, are considered a green alternative to chemical treatments often used in the fight against biofilms' formation in food-related environments [2]. Such enzymes do not have toxic effects and are used both alone and as part of the industrial detergents' composition to improve their cleaning efficacy [64–66].

Proteases are a class of enzymes that catalyzes the cleavage of proteins' peptide bonds. Although they are produced by all living organisms, microbial proteolytic enzymes are preferred over animal or plant origin proteases. The most commonly used source of bacterial proteases is represented by those produced by the genus *Bacillus* since they have remarkable properties such as tolerance to extreme temperatures, large pH domain, organic solvents, detergents, and oxidizing compounds [67]. Given their low substrate specificity, extracellularly produced proteases were shown to be more effective in degrading organic-based aging biofilms compared to amylases [68]. Combinations of a buffer that contained surfactants and dispersing and chelating agents with serine proteases and polysaccharidases were shown to be efficient in removing the biofilms formed by *B. cereus* and *P. fluorescens*, respectively, on stainless

**223**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

steel slides by the cleaning-in-place procedure [69]. Purified alkaline proteases from *B. subtilis* were reported to degrade biofilms produced by both *P. mendocina* and *E. coli* within 10 minutes [70]. Mold-origin proteases, such as proteinase K, were proved to be effective agents against biofilms formed by *L. monocytogenes* when used either alone or in combination with other biofilms' inhibitors. In a study, proteinase K was capable of complete dispersion of *L. monocytogenes* biofilms grown for 72 h on both plastic and stainless steel surfaces at concentrations above 25 μg/mL. The same study also emphasized the synergistic effect between DNases and proteinase K regarding

Polysaccharide-hydrolyzing enzymes were indicated to remove the biofilms formed by *Staphylococcus* spp. and *Pseudomonas* spp. on steel and polypropylene substrata. However, these enzymes did not exhibit a significant bactericidal effect, so they were combined with oxidoreductases for an improved performance [72]. Experimental studies showed that cellulase in conjunction with cetyltrimethylammonium bromide had the capacity of removing 100% of the *S. enterica* mature biofilm at the phase of irreversible attachment. This finding suggests an alternative

strategy for removing *Salmonella* biofilms in meat processing facilities [73].

Plant essential oils (EOs) are rich in phytochemical compounds, which are secondary metabolites produced by plants as defense mechanism against pathogens [74]. Regarding microbial inactivation, EOs have been reported to mainly affect the cellular membrane by permeabilization [75]. This leads to the disruption of vital cellular processes, including energy production, membrane transport, and meta-

Studies evaluating the potential of EOs as disinfectants were conducted. Leonard et al. [77] assessed the bioactivity of *Syzygium aromaticum* (clove), *Mentha spicata* (spearmint), *Lippia rehmannii*, *Cymbopogon citratus* (lemongrass) EOs, and their major components on the listerial biofilm. The assessment revealed that *M. spicata* and *S. aromaticum* EOs inhibited the growth of listerial biofilm, while, surprisingly, in the presence of their main compounds alone, namely R-(−) carvone and eugenol, respectively, the biofilm biomass increased. Similar phenomenon was previously noticed by [78] in the case of α-pinene, 1,8-cineole, (+)-limonene, linalool, and geranyl acetate, with researchers arguing that bacterial cells in biofilms have a reduced metabolic activity, which make them more resistant to deleterious agents. These results suggest that antimicrobial activity of EOs is rather due to the synergism among the chemical substances that compose them, than due to an individual component's activity. On the other hand, a disinfectant solution based on *Cymbopogon citratus* and *Cymbopogon nardus* EOs was reported to completely reduce the number of *L. monocytogenes* stainless steel surface-adhered cells residing in a

Thyme EO has proven antimicrobial properties [80]. In terms of biofilm inhibition capacity, this EO was shown to inhibit significantly the biofilm formed by *B. cereus* [81] and biofilms formed by other food-related pathogens, including *S. aureus* and *E. coli* [82, 83]. Thymol and carvacrol are principal constituents of thyme oil [84], and their potential regarding biofilm inhibition is intensively studied. Surfactant-encapsulated carvacrol was effective against biofilms produced by *E. coli* O157:H7 and *L. monocytogenes* on stainless steel coupons [85]. This natural biocide was also shown to control a dual-species biofilm formed by *S. aureus* and *S. enterica* at quasi-steady state [86]. However, scientists emphasized that carvacrol concentration should be seriously considered when used to combat strong biofilm producers, such as *S. aureus* strains isolated from food-contact surfaces, since low

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

*L. monocytogenes*-established biofilm dispersion [71].

**4.3 Using essential oils against biofilms**

240 h biofilm after 60 min of interaction [79].

bolic regulatory functions [76].

### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

Bower et al. [62].

*sonorensis* MT93 [63].

addressed to find effective solutions that can prevent biofilm formation or eliminate the already formed one. Biocontrol of biofilms by using bacteriocins, disruptive enzymes, essential oils, or bacteriophages is gaining importance, as well as using nanoemulsions and nanoparticles. These new methods are promising strategies

Bacteriocins are antimicrobial peptides ribosomally produced by an extensive range of bacteria to inhibit or kill competing microorganisms in a micro-ecological system [52, 53]. The most studied bacteriocin and the only one allowed presently as food-grade additive is nisin, a lantibiotic with proven effects against many Grampositive bacteria including foodborne pathogens [54]. This bacteriocin was shown to penetrate the biofilm formed by *S. aureus* and permeate the sessile bacterial cells by real-time monitoring [55]. Moreover, nisin and its bioengineered derivatives were able to enhance the capability of conventional antibiotics such as chloramphenicol of decreasing *S. aureus* biofilm viability [56]. Nevertheless, a study assessing the effect of neutral electrolyzed water and nisin and their combination against listerial biofilm on glass and stainless steel surfaces indicated the potency of this bacteriocin to improve the efficacy of sanitizers used in food industry [57]. Nisin was also indicated to be effective against biofilms formed by Gram-negative bacteria such as *Salmonella* typhimurium when combined with P22 phage and EDTA, a

synergistic combination that reduced 70% of the mature biofilm [58].

**4.2 Disruptive enzymes for fighting against biofilms**

detergents' composition to improve their cleaning efficacy [64–66].

Another way to prevent biofilms development is represented by the adsorption of these bioactive compounds on the surfaces that come into contact with foods [59]. In this case, Nisaplin adsorbed to three types of food-contact surfaces commonly encountered in food processing plants, namely stainless steel, polyethylene terephthalate (PET), and rubber, reduced the adhesion ability of food-isolated *L. monocytogenes* strains [60]. Other studies showing the efficacy of nisin in preventing surface colonization by *L. monocytogenes* were conducted by Daeschel et al. [61] and

A bacteriocin found to markedly inhibit the biofilm formed by *S. aureus* is sonorensis, a member of the heterocycloanthracin subfamily produced by *Bacillus* 

Disruptive enzymes, such as proteases, glycosidases, amylases, cellulases, or DNAses, are considered a green alternative to chemical treatments often used in the fight against biofilms' formation in food-related environments [2]. Such enzymes do not have toxic effects and are used both alone and as part of the industrial

Proteases are a class of enzymes that catalyzes the cleavage of proteins' peptide bonds. Although they are produced by all living organisms, microbial proteolytic enzymes are preferred over animal or plant origin proteases. The most commonly used source of bacterial proteases is represented by those produced by the genus *Bacillus* since they have remarkable properties such as tolerance to extreme temperatures, large pH domain, organic solvents, detergents, and oxidizing compounds [67]. Given their low substrate specificity, extracellularly produced proteases were shown to be more effective in degrading organic-based aging biofilms compared to amylases [68]. Combinations of a buffer that contained surfactants and dispersing and chelating agents with serine proteases and polysaccharidases were shown to be efficient in removing the biofilms formed by *B. cereus* and *P. fluorescens*, respectively, on stainless

with remarkable results in the fight against biofilms.

**4.1 Bacteriocins used to control biofilms**

**222**

steel slides by the cleaning-in-place procedure [69]. Purified alkaline proteases from *B. subtilis* were reported to degrade biofilms produced by both *P. mendocina* and *E. coli* within 10 minutes [70]. Mold-origin proteases, such as proteinase K, were proved to be effective agents against biofilms formed by *L. monocytogenes* when used either alone or in combination with other biofilms' inhibitors. In a study, proteinase K was capable of complete dispersion of *L. monocytogenes* biofilms grown for 72 h on both plastic and stainless steel surfaces at concentrations above 25 μg/mL. The same study also emphasized the synergistic effect between DNases and proteinase K regarding *L. monocytogenes*-established biofilm dispersion [71].

Polysaccharide-hydrolyzing enzymes were indicated to remove the biofilms formed by *Staphylococcus* spp. and *Pseudomonas* spp. on steel and polypropylene substrata. However, these enzymes did not exhibit a significant bactericidal effect, so they were combined with oxidoreductases for an improved performance [72]. Experimental studies showed that cellulase in conjunction with cetyltrimethylammonium bromide had the capacity of removing 100% of the *S. enterica* mature biofilm at the phase of irreversible attachment. This finding suggests an alternative strategy for removing *Salmonella* biofilms in meat processing facilities [73].

## **4.3 Using essential oils against biofilms**

Plant essential oils (EOs) are rich in phytochemical compounds, which are secondary metabolites produced by plants as defense mechanism against pathogens [74]. Regarding microbial inactivation, EOs have been reported to mainly affect the cellular membrane by permeabilization [75]. This leads to the disruption of vital cellular processes, including energy production, membrane transport, and metabolic regulatory functions [76].

Studies evaluating the potential of EOs as disinfectants were conducted. Leonard et al. [77] assessed the bioactivity of *Syzygium aromaticum* (clove), *Mentha spicata* (spearmint), *Lippia rehmannii*, *Cymbopogon citratus* (lemongrass) EOs, and their major components on the listerial biofilm. The assessment revealed that *M. spicata* and *S. aromaticum* EOs inhibited the growth of listerial biofilm, while, surprisingly, in the presence of their main compounds alone, namely R-(−) carvone and eugenol, respectively, the biofilm biomass increased. Similar phenomenon was previously noticed by [78] in the case of α-pinene, 1,8-cineole, (+)-limonene, linalool, and geranyl acetate, with researchers arguing that bacterial cells in biofilms have a reduced metabolic activity, which make them more resistant to deleterious agents. These results suggest that antimicrobial activity of EOs is rather due to the synergism among the chemical substances that compose them, than due to an individual component's activity. On the other hand, a disinfectant solution based on *Cymbopogon citratus* and *Cymbopogon nardus* EOs was reported to completely reduce the number of *L. monocytogenes* stainless steel surface-adhered cells residing in a 240 h biofilm after 60 min of interaction [79].

Thyme EO has proven antimicrobial properties [80]. In terms of biofilm inhibition capacity, this EO was shown to inhibit significantly the biofilm formed by *B. cereus* [81] and biofilms formed by other food-related pathogens, including *S. aureus* and *E. coli* [82, 83]. Thymol and carvacrol are principal constituents of thyme oil [84], and their potential regarding biofilm inhibition is intensively studied. Surfactant-encapsulated carvacrol was effective against biofilms produced by *E. coli* O157:H7 and *L. monocytogenes* on stainless steel coupons [85]. This natural biocide was also shown to control a dual-species biofilm formed by *S. aureus* and *S. enterica* at quasi-steady state [86]. However, scientists emphasized that carvacrol concentration should be seriously considered when used to combat strong biofilm producers, such as *S. aureus* strains isolated from food-contact surfaces, since low

concentrations may exhibit an inductive effect. In the case of the biofilm formed by *Salmonella* typhimurium on stainless steel surfaces, exposure to thymol resulted in a more pronounced decrease in the biofilm mass compared to exposure to carvacrol or eugenol [87]. Moreover, these compounds enhanced the susceptibility of this pathogen to the treatments with antibiotics such as nalidixic acid [88].

Eugenol is a phytochemical compound preponderantly found in aromatic plants [89]. Interestingly, a study showed that this substance was able to inhibit the intracellular signaling pathway called quorum sensing in the case of biofilms formed by methicillin-resistant *S. aureus* strains isolated from food handlers. This mechanism has an important role in the host colonization, biofilm development, and defense strategies against harmful agents, allowing bacterial cells to act as social communities [90]. EOs of bay, clove, pimento berry, and their major constituent, eugenol, were proved to inhibit significantly the biofilm formed by *E. coli* O157:H7. The antibiofilm activity was assigned to the benzene ring of eugenol. Moreover, eugenol led to the downregulation of genes associated with the biofilm formation, attachment, and effacement phenotype, such as curli, fimbriae, and toxin genes [91].

### **4.4 Fighting against biofilms with bacteriophages**

Bacteriophages are viruses that infect bacterial cells. They use the genetic machinery of their host cells to replicate, killing bacteria when reaching a sufficiently high number to produce lysis [92]. They are abundantly encountered anywhere host bacteria live [93] and, therefore, their potential is presently harnessed as natural antimicrobial agents to control pathogenic bacteria in food products and food-related environments [94]. One of the bacteriophages' applications that is intensively explored targets biofilm-forming bacteria that are relevant for food industry, including *L. monocytogenes, S. aureus, E. coli, B. cereus*, and *S. enterica*. However, the success of this approach in fighting biofilms depends on a series of factors such as composition and structure of biofilms, biofilms' maturity, and physiological state of bacterial host residing within biofilms, concentration of bacterial host, or extracellular matrix [95].

Although it is generally thought that biofilms confer resistance to bacteriophages, these bacterial predators developed several mechanisms to destroy bacteria communities. Once they reach the EPS (extracellular polymeric substances) producing host, they start to replicate, resulting in an increased number and, implicitly, in a progressive degradation of the biofilms and prevention of their regeneration. Bacteriophages can also express or induce the expression from within host genome of depolymerizing enzymes that degrade EPS. Nevertheless, they can also infect persister cells, which are dormant variants of regular bacterial cells that are highly resistant to antibiotics. In this case, the lysis process is triggered once persister bacteria are reactivated [96].

Scientists [97] reported the ability of a bred phage to reduce L-form biofilms formed by *L. monocytogenes* on stainless steel surfaces. This bacteriophage was as effective as lactic acid (130 ppm) in the eradication of preformed L-form biofilms. P100 phage treatment was also shown to reduce the number of *L. monocytogenes* cells under biofilm conditions on stainless steel coupon surface regardless of serotype [98]. The potency of three bacteriophages, namely LiMN4L, LiMN4p, and LiMN17, used as a cocktail or individually at ~9 log10 PFU/mL was evaluated to inactivate *L. monocytogenes* cells residing within 7-day biofilms strongly adhered to clean or fish broth-coated stainless steel coupons and dislodged biofilm cells [99]. These phages exhibited a higher efficiency in the case of dislodged cells compared to intact biofilms when applied for short periods of time. Therefore, for high efficiency, short-term phage treatments in fish processing environments may require

**225**

**Table 2.**

*Biofilms Formed by Pathogens in Food and Food Processing Environments*

suggested as effective antibiofilm agent in food industry [101].

film activity being an alternative to conventional methods.

food matrix [105–107].

eradication of already formed ones [108].

**Nanoemulsion Particle size,** 

Trans-CA >100

EO of *Citrus medica* L. var. *sarcodactylis*

EO of *Cymbopogon flexuosus* (lemongrass)

**nm**

<100

Linalool 10.9 ± 0.1 *S.* typhimurium

*Antibiofilm activity of essential oil (EO) nanoemulsions.*

**4.5 Nanotechnology-based antimicrobials used to control biofilms**

Currently, controlling biofilm formation by nanotechnology-based antimicrobials is of industrial interest, nanoemulsions and nanoparticles (NPs) with antibio-

Recently, some studies made on model system (polystyrene well plates) and real systems (fresh pineapple, tofu, and lettuce) indicated that nanoemulsions of EOs have significantly higher antibiofilm activity compared to pure EOs (**Table 2**). Antimicrobial efficacy of nanoemulsions is dependent on the droplet size and electrical properties of nanoemulsions [102, 103], nature of bacteria [75, 104], and

Nanoparticles (NPs) can be used for both inhibition of biofilm formation and

it was demonstrated that the inorganic capsules can protect the natural products with antimicrobial activity [109]. In this respect, cinnamaldehyde-encapsulated chitosan nanoparticles, garlic-silver NPs, and "tree of tee" oil NPs were used to combat biofilm formation by *P. aeruginosa* on polystyrene well plates and glass pieces [110–112]. Meanwhile, the biofilm formed by *S. aureus* on glass slide was inhibited by

applying gold NPs with EO of *Nigella sativa* [113] and garlic-silver NPs [111].

78.46 ± 0.51 *P. aeruginosa* (PA01)

In the last period, NPs with natural compounds gained increased interest because

**Biofilm-forming bacteria**

and *S. aureus* (ATCC 29213)

*P. aeruginosa* (CMCC 10104), *S.* typhimurium and *S. aureus*

(ATCC 1331)

73 *S. aureus* Inhibit the ability of

**Mode of action Ref.**

[185]

[186]

[187]

[107]

bacteria to attach to surfaces

Reduce the adhesion of pathogenic bacteria to surfaces

Membrane disruption by destabilization of lipids

Cell membrane integrity disruption

prior processes aiming at disrupting the biofilms [99]. The ability of *Salmonella* spp. to develop biofilms was shown to depend on the attachment surface types that may be encountered in chicken slaughterhouses. With regard to this, surfaces such as glass and stainless steel favored the formation of *Salmonella* biofilms, while polyvinyl chloride surface sustained less the development of them. The antibiofilm activity of a pool of bacteriophages isolated from hospital and poultry wastewater was concentrated at 3 h of action for all types of surfaces. Curiously, biofilms attached to the glass surface were resistant to a 6-h treatment. Bacteriophages were able to degrade the glass-attached biofilms after 9 h of interaction [100]. A bacteriophage BPECO 19 was evaluated as possible inhibitor of a three *E. coli* O157:H7 strain biofilm grown on both abiotic (stainless steel, rubber, and minimum biofilm eradication concentration device) and biotic (lettuce leaves) surfaces. This bacteriophage showed great biofilm inhibition activity on all the tested surfaces, being

*DOI: http://dx.doi.org/10.5772/intechopen.90176*

### *Biofilms Formed by Pathogens in Food and Food Processing Environments DOI: http://dx.doi.org/10.5772/intechopen.90176*

*Bacterial Biofilms*

concentrations may exhibit an inductive effect. In the case of the biofilm formed by *Salmonella* typhimurium on stainless steel surfaces, exposure to thymol resulted in a more pronounced decrease in the biofilm mass compared to exposure to carvacrol or eugenol [87]. Moreover, these compounds enhanced the susceptibility of this

Eugenol is a phytochemical compound preponderantly found in aromatic plants [89]. Interestingly, a study showed that this substance was able to inhibit the intracellular signaling pathway called quorum sensing in the case of biofilms formed by methicillin-resistant *S. aureus* strains isolated from food handlers. This mechanism has an important role in the host colonization, biofilm development, and defense strategies against harmful agents, allowing bacterial cells to act as social communities [90]. EOs of bay, clove, pimento berry, and their major constituent, eugenol, were proved to inhibit significantly the biofilm formed by *E. coli* O157:H7. The antibiofilm activity was assigned to the benzene ring of eugenol. Moreover, eugenol led to the downregulation of genes associated with the biofilm formation, attachment, and effacement phenotype, such as curli, fimbriae, and toxin genes [91].

Bacteriophages are viruses that infect bacterial cells. They use the genetic machinery of their host cells to replicate, killing bacteria when reaching a sufficiently high number to produce lysis [92]. They are abundantly encountered anywhere host bacteria live [93] and, therefore, their potential is presently harnessed as natural antimicrobial agents to control pathogenic bacteria in food products and food-related environments [94]. One of the bacteriophages' applications that is intensively explored targets biofilm-forming bacteria that are relevant for food industry, including *L. monocytogenes, S. aureus, E. coli, B. cereus*, and *S. enterica*. However, the success of this approach in fighting biofilms depends on a series of factors such as composition and structure of biofilms, biofilms' maturity, and physiological state of bacterial host residing within biofilms, concentration of

Although it is generally thought that biofilms confer resistance to bacteriophages, these bacterial predators developed several mechanisms to destroy bacteria communities. Once they reach the EPS (extracellular polymeric substances) producing host, they start to replicate, resulting in an increased number and, implicitly, in a progressive degradation of the biofilms and prevention of their regeneration. Bacteriophages can also express or induce the expression from within host genome of depolymerizing enzymes that degrade EPS. Nevertheless, they can also infect persister cells, which are dormant variants of regular bacterial cells that are highly resistant to antibiotics. In this case, the lysis process is triggered once

Scientists [97] reported the ability of a bred phage to reduce L-form biofilms formed by *L. monocytogenes* on stainless steel surfaces. This bacteriophage was as effective as lactic acid (130 ppm) in the eradication of preformed L-form biofilms. P100 phage treatment was also shown to reduce the number of *L. monocytogenes* cells under biofilm conditions on stainless steel coupon surface regardless of serotype [98]. The potency of three bacteriophages, namely LiMN4L, LiMN4p, and LiMN17, used as a cocktail or individually at ~9 log10 PFU/mL was evaluated to inactivate *L. monocytogenes* cells residing within 7-day biofilms strongly adhered to clean or fish broth-coated stainless steel coupons and dislodged biofilm cells [99]. These phages exhibited a higher efficiency in the case of dislodged cells compared to intact biofilms when applied for short periods of time. Therefore, for high efficiency, short-term phage treatments in fish processing environments may require

pathogen to the treatments with antibiotics such as nalidixic acid [88].

**4.4 Fighting against biofilms with bacteriophages**

bacterial host, or extracellular matrix [95].

persister bacteria are reactivated [96].

**224**

prior processes aiming at disrupting the biofilms [99]. The ability of *Salmonella* spp. to develop biofilms was shown to depend on the attachment surface types that may be encountered in chicken slaughterhouses. With regard to this, surfaces such as glass and stainless steel favored the formation of *Salmonella* biofilms, while polyvinyl chloride surface sustained less the development of them. The antibiofilm activity of a pool of bacteriophages isolated from hospital and poultry wastewater was concentrated at 3 h of action for all types of surfaces. Curiously, biofilms attached to the glass surface were resistant to a 6-h treatment. Bacteriophages were able to degrade the glass-attached biofilms after 9 h of interaction [100]. A bacteriophage BPECO 19 was evaluated as possible inhibitor of a three *E. coli* O157:H7 strain biofilm grown on both abiotic (stainless steel, rubber, and minimum biofilm eradication concentration device) and biotic (lettuce leaves) surfaces. This bacteriophage showed great biofilm inhibition activity on all the tested surfaces, being suggested as effective antibiofilm agent in food industry [101].
