Study of the Resistance of Staphylococcus aureus Biofilm, Biofilm-Detached Cells, and Planktonic Cells to Microencapsulated Carvacrol Used Alone or Combined with Low-pH Treatment.

Microbial biofilms pose severe problems in the medical field and food industry, as they are the cause of many serious infections and food-borne diseases. The extreme biofilms' resistance to conventional anti-microbial treatments presents a major challenge to their elimination. In this study, the difference in resistance between Staphylococcus aureus DSMZ 12463 biofilms, biofilm-detached cells, and planktonic cells against microcapsules containing carvacrol was assessed. The antimicrobial/antibiofilm activity of low pH disinfection medium containing the microencapsulated carvacrol was also studied. In addition, the effect of low pH on the in vitro carvacrol release from microcapsules was investigated. The minimum inhibitory concentration of microencapsulated carvacrol was 0.625 mg mL-1. The results showed that biofilms exhibited greater resistance to microencapsulated carvacrol than the biofilm-detached cells and planktonic cells. Low pH treatment alone, by hydrochloric acid addition, showed no bactericidal effect on any of the three states of S. aureus strain. However, microencapsulated carvacrol was able to significantly reduce the planktonic cells and biofilm-detached cells below the detection limit (no bacterial counts), and the biofilm by approximatively 3 log CFU mL-1. In addition, results showed that microencapsulated carvacrol combined with low pH treatment reduced biofilm by more than 5 log CFU mL-1. Thus, the use of microencapsulated carvacrol in acidic environment could be a promising approach to combat biofilms from abiotic surfaces.

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol.

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04-6.05 log CFU cm-2) than the mutant strains (2.30-5.48 log CFU cm-2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58-2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58-2.90) than the ones in TSB (2.02-3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

Nanoemulsion-Based Multilayer Films for Ground Beef Preservation: Antimicrobial Activity and Physicochemical Properties.

This study aimed to improve the physical, mechanical, and biological properties of a monolayer pectin (P) film containing nanoemulsified trans-Cinnamaldehyde (TC) by incorporating it between inner and outer layers of ethylcellulose (EC). The nanoemulsion had an average size of 103.93 nm and a zeta potential of -46 mV. The addition of the nanoemulsion increased the opacity of the film, reduced its moisture absorption capacity, and improved its antimicrobial activity. However, the tensile strength and elongation at break of the pectin films decreased after the incorporation of nanoemulsions. Multilayer films (EC/P/EC) showed a higher resistance to breaking and better extensibility compared to monolayer films. The antimicrobial activity of both mono and multilayer films was effective in inhibiting the growth of foodborne bacteria during storage of ground beef patties at 8 °C for 10 days. This study suggests that biodegradable antimicrobial multilayer packaging films can be effectively designed and applied in the food packaging industry.

Pepsin and Trypsin Treatment Combined with Carvacrol: An Efficient Strategy to Fight Pseudomonas aeruginosa and Enterococcus faecalis Biofilms.

Biofilms consist of microbial communities enclosed in a self-produced extracellular matrix which is mainly responsible of biofilm virulence. Targeting this matrix could be an effective strategy to control biofilms. In this work, we examined the efficacy of two proteolytic enzymes, pepsin and trypsin, to degrade P. aeruginosa and E. faecalis biofilms and their synergistic effect when combined with carvacrol. The minimum dispersive concentrations (MDCs) and the contact times of enzymes, as well as the minimal inhibitory concentrations (MICs) and contact times of carvacrol, were determined against biofilms grown on polystyrene surfaces. For biofilms grown on stainless steel surfaces, the combined pepsin or trypsin with carvacrol treatment showed more significant reduction of both biofilms compared with carvacrol treatment alone. This reduction was more substantial after sequential treatment of both enzymes, followed by carvacrol with the greatest reduction of 4.7 log CFU mL−1 (p < 0.05) for P. aeruginosa biofilm and 3.3 log CFU mL−1 (p < 0.05) for E. faecalis biofilm. Such improved efficiency was also obvious in the epifluorescence microscopy analysis. These findings demonstrate that the combined effect of the protease-dispersing activity and the carvacrol antimicrobial activity could be a prospective approach for controlling P. aeruginosa and E. faecalis biofilms.

Dynamic Salmonella Enteritidis biofilms development under different flow conditions and their removal using nanoencapsulated thymol.

In food industries, microbial contaminations are difficult to control due to the recurrent formation of biofilms that hinders antimicrobials penetration and efficiency. An understanding of Salmonella Enteritidis biofilms behavior under flow conditions is a key to develop efficient preventive and control strategies. S. Enteritidis biofilms displayed 5.96, 6.28 and 6.80 log CFU cm-2 under 0.006 cm s-1, 0.045 cm s-1, and 0.087 cm s-1 flow velocities, respectively. Biofilms exposed to higher nutrient conditions under greater flow rates, induced significantly more biofilm biomass. To control biofilms, the disinfection efficiency of thymol (THY) was assessed under dynamic conditions by encapsulation it into two types of nanocapsules: monolayer (ML) nanocapsules prepared with a single carrier material (maltodextrin), and layer-by-layer (LBL) nanocapsules prepared by combining two carrier materials (maltodextrin and pectin). A combined mixture of ML and LBL nanocapsules at ½ their minimal inhibitory concentrations induced 99.99% eradication of biofilms developed under the highest flow conditions, after 5 h. ML nanocapsules decreased significantly bacterial counts during the first 0.5 h, while LBL nanocapsules eliminated the remaining bacterial cells and ensured a protection from bacterial contamination for up to 5 h by releasing THY in a sustained manner over time due to the thicker shell wall structure.

Hurdle technology based on the use of microencapsulated pepsin, trypsin and carvacrol to eradicate Pseudomonas aeruginosa and Enterococcus faecalis biofilms.

The biofilm lifestyle plays a major role in the resistance and virulence of Pseudomonas aeruginosa and Enterococcus faecalis. In this study, two microencapsulated proteases (pepsin ME-PEP and trypsin ME-TRYP) were evaluated for their biofilm dispersal activity and their synergistic effect with microencapsulated carvacrol (ME-CARV). Spray-drying was used to protect enzymes and essential oil and enhance their activities. Cell count analysis proved the synergistic activity of enzymes and carvacrol treatment as biofilms were further reduced after combined treatment in comparison to ME-CARV or enzymes alone. Furthermore, results showed that sequential treatment in the order ME-TRYP - ME-PEP - ME-CARV resulted in more efficient biofilm removal with a maximum reduction of 5 log CFU mL-1 for P. aeruginosa and 4 log CFU mL-1 for E. faecalis. This study proposes that the combination of microencapsulated proteases with ME-CARV could be useful for the effective control of P. aeruginosa and E. faecalis biofilms.

Microencapsulation of carvacrol as an efficient tool to fight Pseudomonas aeruginosa and Enterococcus faecalis biofilms.

Biofilms are involved in serious problems in medical and food sectors due to their contribution to numerous severe chronic infections and foodborne diseases. The high resistance of biofilms to antimicrobial agents makes their removal as a big challenge. In this study, spray-drying was used to develop microcapsules containing carvacrol, a natural antimicrobial agent, to enhance its activity against P. aeruginosa and E. faecalis biofilms. The physicochemical properties and microscopic morphology of the realized capsules and cells were characterized. The minimum inhibitory concentration of encapsulated carvacrol (E-CARV) (1.25 mg mL-1) was 4-times lower than that of free carvacrol (F-CARV) (5 mg mL-1) against P. aeruginosa, while it remained the same against E. faecalis (0.625 mg mL-1). E-CARV was able to reduce biofilm below the detection limit for P. aeruginosa and by 5.5 log CFU ml-1 for E. faecalis after 15 min of treatment. Results also showed that F-CARV and E-CARV destabilize the bacterial cell membrane leading to cell death. These results indicate that carvacrol exhibited a strong antimicrobial effect against both bacterial biofilms. In addition, spray-drying could be used as an effective tool to enhance the antibiofilm activity of carvacrol, while reducing the concentrations required for disinfection of abiotic surfaces.

Hurdle technology using encapsulated enzymes and essential oils to fight bacterial biofilms.

Biofilm formation on abiotic surfaces has become a major public health concern because of the serious problems they can cause in various fields. Biofilm cells are extremely resistant to stressful conditions, because of their complex structure impedes antimicrobial penetration to deep-seated cells. The increased resistance of biofilm to currently applied control strategies underscores the urgent need for new alternative and/or supplemental eradication approaches. The combination of two or more methods, known as Hurdle technology, offers an excellent option for the highly effective control of biofilms. In this perspective, the use of functional enzymes combined with biosourced antimicrobial such as essential oil (EO) is a promising alternative anti-biofilm approach. However, these natural antibiofilm agents can be damaged by severe environmental conditions and lose their activity. The microencapsulation of enzymes and EOs is a promising new technology for enhancing their stability and improving their biological activity. This review article highlights the problems related to biofilm in various fields, and the use of encapsulated enzymes with essential oils as antibiofilm agents. KEY POINTS: • Problems associated with biofilms in the food and medical sectors and their subsequent risks on health and food quality. • Hurdle technology using enzymes and essential oils is a promising strategy for an efficient biofilms control. • The microencapsulation of enzymes and essential oils ensures their stability and improves their biological activities.

Bioinformatics Modelling and Metabolic Engineering of the Branched Chain Amino Acid Pathway for Specific Production of Mycosubtilin Isoforms in Bacillus subtilis.

Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.

Essential oils and their active components applied as: free, encapsulated and in hurdle technology to fight microbial contaminations. A review.

Microbial contaminations are responsible for many chronic, healthcare, persistent microbial infections and illnesses in the food sector, therefore their control is an important public health challenge. Over the past few years, essential oils (EOs) have emerged as interesting alternatives to synthetic antimicrobials as they are biodegradable, extracted from natural sources and potent antimicrobials. Through their multiple mechanisms of actions and target sites, no microbial resistance has been developed against them till present. Although extensive documentation has been reported on the antimicrobial activity of EOs, comparisons between the use of whole EOs or their active components alone for an antimicrobial treatment are less abundant. It is also essential to have a good knowledge about EOs to be used as alternatives to the conventional antimicrobial products such as chemical disinfectants. Moreover, it is important to focus not only on planktonic vegetative microorganisms, but to study also the effect on more resistant forms like spores and biofilms. The present article reviews the current knowledge on the mechanisms of antimicrobial activities of EOs and their active components on microorganisms in different forms. Additionally, in this review, the ultimate advantages of encapsulating EOs or combining them with other hurdles for enhanced antimicrobial treatments are discussed.

Cold plasma surface treatments to prevent biofilm formation in food industries and medical sectors.

Environmental conditions in food and medical fields enable the bacteria to attach and grow on surfaces leading to resistant bacterial biofilm formation. Indeed, the first step in biofilm formation is the bacterial irreversible adhesion. Controlling and inhibiting this adhesion is a passive approach to fight against biofilm development. This strategy is an interesting path in the inhibition of biofilm formation since it targets the first step of biofilm development. Those pathogenic structures are responsible for several foodborne diseases and nosocomial infections. Therefore, to face this public health threat, researchers employed cold plasma technologies in coating development. In this review, the different factors influencing the bacterial adhesion to a substrate are outlined. The goal is to present the passive coating strategies aiming to prevent biofilm formation via cold plasma treatments, highlighting antiadhesive elaborated surfaces. General aspects of surface treatment, including physico-chemical modification and application of cold plasma technologies, were also presented. KEY POINTS: • Factors surrounding pathogenic bacteria influence biofilm development. • Controlling bacterial adhesion prevents biofilm formation. • Materials can be coated via cold plasma to inhibit bacterial adhesion.

Water-Soluble Ruthenium (II) Complex Derived From Optically Pure Limonene and Its Microencapsulation Are Efficient Tools Against Bacterial Food Pathogen Biofilms: Escherichia coli, Staphylococcus aureus, Enteroccocus faecalis, and Listeria monocytogenes.

Bioactive aminooxime ligands based on optically pure (R)-limonene have been synthesized in two steps. Their ruthenium (II) cationic water-soluble complex was prepared by a reaction between dichloro (para-cymene) ruthenium (II) dimers and aminooxime ligands in a 1:2 molar ratio. Antibacterial and antibiofilm activities of the synthetized complex were assessed against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis. The results revealed that the ruthenium (II) complex has higher antibacterial and antibiofilm activities in comparison with free ligands or the enantiopure (R)-limonene. Moreover, microencapsulation of this complex reduced its cytotoxicity and improved their minimum inhibitory concentration and antibiofilm activity toward the considered bacteria. The ruthenium (II) complex targets the bacterial cell membrane, which leads to rapid leakage of intracellular potassium. Our study suggests that the developed ruthenium (II) complexes could be useful as an alternative to conventional disinfectants.

Preparation and Characterization of Microcapsules Containing Antioxidant Fish Protein Hydrolysates: a New Use of Bycatch in Brazil.

In this study, we evaluated the effect of complexation and microencapsulation with pea protein on the antioxidant activity of protein hydrolysates from bycatch in Brazil. The zeta potential values of complexes changed from negative to positive with the increase of pea protein as a result of positively charged complexes formation. The increase in the ratio of pea protein/hydrolysates also resulted in increased turbidity in all samples. Particle size measurements indicated that the complexes tended to form larger aggregates (ranged from 61.5 ± 1.7 µm to 183 ± 2.8 µm). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the pea protein/fish protein hydrolysate complexes was higher than that of the protein hydrolysates alone. Moreover, increasing levels of pea protein did not affect the antioxidant activity of fish protein hydrolysates. The complexes of the Paralonchurus brasiliensis were chosen for the microencapsulation process by spray-drying. The results revealed that spray-drying did not have a significant effect (P > 0.05) on the protein hydrolysate antioxidant activity when they were complexed with pea protein. Thus, this work suggests that the complexation with pea protein and subsequent microencapsulation by spray-drying is an efficient way to protect the biological activity of protein hydrolysates obtained from bycatch. This study provides evidence for the potential use of bycatch from shrimp fisheries as functional ingredients or nutraceuticals.

Cold plasma assisted deposition of organosilicon coatings on stainless steel for prevention of adhesion of Salmonella enterica serovar Enteritidis.

The persistence of Salmonella enterica on abiotic surfaces in hospitals and the agri-food industries leads to several infections worldwide. In this context, this work aimed to study the adhesion of S. Enteritidis on plasma-modified stainless steel to prevent biofilm-associated-infections. Surface modification was achieved by the elaboration of organosilicon coatings from the monomer 1,1,3,3-tetramethyldisiloxane, mixed with oxygen, using a flowing nitrogen microwave post-discharge plasma polymerization technique. The effect of cold plasma parameters on the properties of the coatings, the coated surface topography and S. Enteritidis cell adhesion was studied. The results showed that the surface topography influenced the bacterial adhesion rate. Indeed, rough surfaces did not repel S. Enteritidis since the number of attached cells on these coatings was between 30 ± 4 to 65 ± 4 bacteria per microscopic field. Otherwise, smoother surfaces demonstrated an anti-adhesive character since the number of attached cells was almost nil on these coatings.

Anti-biofilm activity of dodecyltrimethylammonium chloride microcapsules against Salmonella enterica serovar Enteritidis and Staphylococcus aureus.

Dodecyltrimethylammonium chloride (DTAC) was trapped into maltodextrins/pectin spray dried microcapsules to improve its activity against Salmonella enteritidis and Staphylococcus aureus biofilms. Two different microcapsules were prepared: uncomplexed DTAC-microcapsules (UDM), containing DTAC and maltodextrins; and complexed DTAC-microcapsules (CDM) containing DTAC complexed with pectin and maltodextrins. The minimum inhibitory concentrations (MIC) of both free and microencapsulated DTAC were investigated against S. Enteritidis and S. aureus. The MICs of DTAC were significantly lower when encapsulated. CDM treatment resulted in a 2 and 3.2 log reduction in S. aureus and S. Enteritidis biofilm culturable biomass, respectively. Microencapsulation reduced the cytotoxicity of DTAC by up to 32-fold. Free DTAC and CDM targeted the cell membrane resulting in the leakage of the intracellular molecules and subsequent cell death. The development of DTAC microcapsules reduced the amount of DTAC required to maintain the high standards of cleanliness and hygiene required in the food processing industries.

Conditions of nisin production by Lactococcus lactis subsp. lactis and its main uses as a food preservative.

Nisin is a small peptide produced by Lactococcus lactis ssp lactis that is currently industrially produced. This preservative is often used for growth prevention of pathogenic bacteria contaminating the food products. However, the use of nisin as a food preservative is limited by its low production during fermentation. This low production is mainly attributed to the multitude of parameters influencing the fermentation progress such as bacterial cells activity, growth medium composition (namely carbon and nitrogen sources), pH, ionic strength, temperature, and aeration. This review article focuses on the main parameters that affect nisin production by Lactococcus lactis bacteria. Moreover, nisin applications as a food preservative and the main strategies generally used are also discussed.

Qishta-A Lebanese Heat Concentrated Dairy Product Characteristics and Production Procedures.

This study aims at exploring the chemical composition of a traditional Lebanese dairy product known as Qishta, describing the process of how to prepare it and understanding the mechanisms leading to its formation. The process of making Qishta can be divided into two phases: a hot phase during which milk is heated in a stainless-steel large shallow vessel, and a cold phase consisting of draining, cooling and packaging. According to milk temperature, two reaction zones were identified: zone A with an average temperature of 100 °C, and zone B with an average temperature of 60 °C. The results showed that Qishta had a moisture, fat, protein, lactose and ash content of 68%, 11.7%, 12.1%, 5.4% and 1.6%, respectively. Our findings showed that Qishta is a lipoprotein product having an equal amount of fat and proteins (≈12%); this composition is almost similar to that of Ricotta cheese made from whole milk. In addition, our results assert that the interactions between caseins and whey proteins lead to gel formation. Milk initial fat percentage had a significant effect on Qishta production. The highest yields were obtained when the initial fat percentage was 3.6% (182.5 g of Qishta).

Characterization of Probiotic Properties of Antifungal Lactobacillus Strains Isolated from Traditional Fermenting Green Olives.

The aim of this work is to characterize the potential probiotic properties of 14 antifungal Lactobacillus strains isolated from traditional fermenting Moroccan green olives. The molecular identification of strains indicated that they are composed of five Lactobacillus brevis, two Lactobacillus pentosus, and seven Lactobacillus plantarum. In combination with bile (0.3%), all the strains showed survival rates (SRs) of 83.19-56.51% at pH 3, while 10 strains showed SRs of 31.67-64.44% at pH 2.5. All the strains demonstrated high tolerance to phenol (0.6%) and produced exopolysaccharides. The autoaggregation, hydrophobicity, antioxidant activities, and surface tension value ranges of the strains were 10.29-41.34%, 15.07-34.67%, 43.11-52.99%, and 36.23-40.27 mN/m, respectively. Bacterial cultures exhibited high antifungal activity against Penicillium sp. The cell-free supernatant (CFS) of the cultures showed important inhibition zones against Candida pelliculosa (18.2-24.85 mm), as well as an antibacterial effect against some gram-positive and gram-negative bacteria (10.1-14.1 mm). The neutralized cell-free supernatant of the cultures displayed considerable inhibitory activity against C. pelliculosa (11.2-16.4 mm). None of the strains showed acquired or horizontally transferable antibiotic resistance or mucin degradation or DNase, hemolytic, or gelatinase activities. Lactobacillus brevis S82, Lactobacillus pentosus S75, and Lactobacillus plantarum S62 showed aminopeptidase, ß-galactosidase, and ß-glucosidase activities, while the other enzymes of API-ZYM were not detected. The results obtained revealed that the selected antifungal Lactobacillus strains are considered suitable candidates for use both as probiotic cultures for human consumption and for starters and as biopreservative cultures in agriculture, food, and pharmaceutical industries.

LABiocin database: A new database designed specifically for Lactic Acid Bacteria bacteriocins.

Bacteriocins from lactic acid bacteria (LAB) are successfully applied as natural alternatives to food preservation and to antibiotics; however, information on these antimicrobial peptides (AMPs) is scattered through the literature and databases. Therefore, we developed the LABiocin database, a specialized database on LAB bacteriocins. The database was stored and compiled using MySQL with NetBeans IDE as the platform. Important data are compiled, including bacteriocin name, class, amino acids and nucleic acid sequences, if available. Target microorganisms, origin, status of the producing strains and their culture conditions and extraction and purification methods are also included in this new database. A phylogenetic tree for the mature peptide bacteriocin sequences has also been created. LABiocin is an interactive database with a user-friendly interface that integrates several tools and services and comprises up to 517 LAB bacteriocins. Besides data searching tools, a BLAST tool was integrated into the database to enable the user to perform a homology search against mature peptide sequences. Users can be linked to other databases that contain additional information, particularly about predicted bacteriocin structure and mechanisms of action. The LABiocin database enables comprehensive functional analysis of this special group of AMPs. This would be useful in food preservation and food safety applications and would also have substantial implications for development of new drugs for medical use. LABiocin database is available at labiocin.net.

Comparative Study on the Impact of Growth Conditions on the Physiology and the Virulence of Pseudomonas aeruginosa Biofilm and Planktonic Cells.

The aim of the present work was to study and compare the effect of growth temperature (20, 30, and 37°C) and surface type (stainless steel and polycarbonate) on the production of virulence factors, such as proteases and siderophores, and the risk of surface contamination associated with Pseudomonas aeruginosa biofilm and planktonic cells. The increase of growth temperature from 20 to 37°C increased (approximately twofold) the electronegative charge and the hydrophobicity of the P. aeruginosa biofilm cell surface. P. aeruginosa biofilm cell adhesion to stainless steel and polycarbonate was 5- and 1.5-fold higher than their planktonic counterparts at 20 and 30°C, respectively. The increase of growth temperature from 20 to 37°C increased the production of proteases (twofold) and siderophores (twofold) and the cytotoxicity (up to 30-fold) against the HeLa cell line in the supernatants of P. aeruginosa planktonic and biofilm cultures. This study also highlighted that biofilm and planktonic P. aeruginosa cells exhibited distinct physiological properties with respect to the production of virulence factors and the cytotoxicity against the Hela cell line. Therefore, effective disinfection procedures should be adapted to inactivate bacteria detached from biofilms.