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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 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.

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.

The present work aimed at studying physiological properties of Pseudomonas aeruginosa cells actively detached from biofilm formed on stainless steel and comparing them with their planktonic counterparts as a function of growth temperature (20 °C and 37 °C). The susceptibility of P. aeruginosa cells to benzalkonium chloride (BAC) was studied. Furthermore, the effect of BAC on the cell membrane integrity and the role of the cell membrane fluidity in the cell-scale-resistance mechanism were investigated. Our results showed that actively detached biofilm cells were more susceptible to BAC treatment than planktonic ones. A greater leakage of intracellular potassium after BAC addition was observed in actively detached biofilm cells, which reflects their membrane vulnerability. The rise of the growth temperature from 20 to 37 °C increased the membrane rigidity of planktonic cells comparatively to their actively detached biofilm ones. Under experimental conditions developed in this work, our data highlighted that actively biofilm-detached and planktonic P. aeruginosa cells have distinguishable phenotypes.

Biofilm formation of Pseudomonas aeruginosa and Staphylococcus aureus on food-contact-surfaces represents a significant risk for the public health. In this context, the present study investigates the relationship between the environmental conditions of biofilm formation and the resistance to disinfectants. Therefore, a static biofilm reactor, called NEC-Biofilm System, was established in order to study the effect of growth temperature (20, 30 and 37°C), and of the surface type (stainless steel and polycarbonate), on biofilm resistance to disinfectants. These conditions were selected to mimic the biofilm formation on abiotic surfaces of food processing industries. The antibiofilm assays were performed on biofilms grown during 24 h. The results showed that the growth temperature influenced significantly the biofilm resistance to disinfectants. These data also revealed that the growth temperature has a significant effect on the biofilm structure of both bacteria. Furthermore, the increase of the biofilm growth temperature increased significantly the algD transcript level in sessile P. aeruginosa cells, whereas the icaA one was not affected in S. aureus cells. Overall, our findings show that the biofilm structure and matrix cannot fully explain the biofilm resistance to disinfectant agents. Nevertheless, it underlines the intimate link between environmental conditions, commonly met in food sectors, and the biofilm resistance to disinfectants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The relationship between the environmental conditions of biofilm formation and resistance to disinfectants was studied. Anti-biofilm assays were performed against biofilms grown at 20, 30 and 37°C on stainless steel and polycarbonate, over 24 and 48 h. A rise in growth temperature increased the resistance of 24 h biofilms to disinfectants containing didecyldimethylammonium chloride and decreased it to a disinfectant containing alkyldimethylbenzylammonium chloride. The increase in growth temperature coupled with an incubation time of 24 h promoted increases in both matrix production and the membrane rigidity of sessile cells. An increase in incubation time also increased both matrix production and the membrane rigidity of sessile cells. Such phenomena resulted in an increased resistance to disinfectants of biofilms grown at 20 and 30°C. The resistance of 48 h biofilms to disinfectants decreased with an increase in growth temperature despite the increase in matrix production and the membrane rigidity of sessile cells.

Two hundred and thirty individual clones of microorganisms were recovered from milk tanks and milking machine surfaces at two distinct farms (Bejaja City, Algeria). Of these clones, 130 were identified as lactic acid bacteria (LAB). In addition Escherichia coli, Salmonella, Staphylococcus aureus and Pseudomonas aeruginosa species were identified in the remaining 100 isolates-spoilage isolate. These isolates were assayed for ability to form biofilms. S. aureus, Lactobacillus brevis strains LB1F2, LB14F1 and LB15F1, and Lactobacillus pentosus strains LB2F2 and LB3F2 were identified as the best biofilm formers. Besides, these LAB isolates were able to produce proteinaceous substances with antagonism against the aforementioned spoilage isolates, when grown in MRS or TSB-YE media. During the screening, L. pentosus LB3F2 exhibited the highest antibacterial activity when grown in TSB-YE medium at 30 °C. Additionally, L. pentosus LB3F2 was able to strongly hamper the adhesion of S. aureus SA3 on abiotic surfaces as polystyrene and stainless steel slides. LAB isolates did not show any hemolytic activity and all of them were sensitive to different families of antibiotic tested. It should be pointed out that LB3F2 isolate was not cytotoxic on the intestinal cells but could stimulate their metabolic activity. This report unveiled the potential of LB1F2, LB14F1, LB15F1, LB2F2, and LB3F2 isolates to be used as natural barrier or competitive exclusion organism in the food processing sector as well as a positive biofilm forming bacteria., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Emerging technology regarding antimicrobial coatings contributes to fighting the challenge of pathogenic bacterial biofilms in medical and agri-food environments. Stainless steel is a material widely used in those fields since it has satisfying mechanical properties, but it, unfortunately, lacks the required bio-functionality, rendering it vulnerable to bacterial adhesion and biofilm formation. Therefore, this review aims to present the coatings developed by employing biocides grafted on stainless steel. It also highlights antimicrobial peptides (AMPs)used to coat stainless steel, particularly nisin, which is commonly accepted as a safe alternative to prevent pathogenic biofilm development. [ABSTRACT FROM AUTHOR]

Following publication of the original article (Khelissa et al. 2017), the authors reported that the legend for Fig. 3 contained an error. Instead of 'Adhesion of planktonic and biofilm-detached Staphylococcus aureus cells on stainless steel and polycarbonate. Cell cultures were grown at 20, 30 and 37 °C, during 24 h (white square) and 48 h (white square). Planktonic cells adhesion on stainless steel (a) and polycarbonate (b). Adhesion of stainless steel-biofilm-detached-cells on stainless steel 24 (c) and polycarbonate-biofilm-detached-cells on polycarbonate (d)', the legend should read 'Adhesion of planktonic and biofilm-detached Staphylococcus aureus cells on stainless steel and polycarbonate. Cell cultures were grown at 20, 30 and 37 °C, during 24 h (black square) and 48 h (white square). Planktonic cells adhesion on stainless steel (a) and polycarbonate (b). Adhesion of stainless steel-biofilm-detached-cells on stainless steel 24 (c) and polycarbonate-biofilm-detached-cells on polycarbonate (d)'. [ABSTRACT FROM AUTHOR]

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, Δ sigB and Δ agrA, 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. • Greater biofilm formation at pH 5.5 and 6.5 in both culture media and food • Nanoencapsulated carvacrol was effective in biofilm removal, especially at pH 5.5. • Mutant L. monocytogenes were more vulnerable in acidic conditions. • Food substrates reduced nanoencapsulated carvacrol's efficacy. [ABSTRACT FROM AUTHOR]