Nisin-based coatings for the prevention of biofilm formation: Surface characterization and antimicrobial assessments

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Cold plasma treatments of materials for preventing bacterial adhesion

Aiming to fight biofilm formation in medical and food fields, many studies were carried out to produce anti-bacterial and anti-adhesive modified surfaces via cold plasma treatments. Among the several materials applied in those fields, stainless steel is a predominant metal used in various application where hygiene is primordial, including food industry and medical sectors (Fouda and Ellithy 2009; Sun et al. 2015). Moreover, titanium alloy is used for dental implants, medical equipment and in food and pharmaceutical manufacturing areas (Agripa and Botef 2019). In addition, polyethylene terephthalate (PET) is a polymer commonly used in biomedical and food applications (Perez-Roldan et al. 2014). Other polymers like polyamide, polydimethylsiloxane (PDMS), silicone and polypropylene can be applied for their physico-chemical properties in those sectors. This section highlights the strategies carried out by researchers to elaborate antiadhesive films by cold plasma treatment on the materials surfaces mentioned above. Fig. 9 summarize the main directions followed in coatings elaboration and Fig. 10 shows the general surface properties modifications after plasma treatment.
Several studies demonstrated that modifying surfaces with hydrophilic and non-charged polymers resulted in reduced cellular, protein, and bacterial attachment on different surface types (Finch 1994; Du et al. 1997; Sofia et al. 1998; Zhang et al. 2001). Indeed, it has been established that surfaces deposited with poly(ethylene glycol) (PEG) are able to reduce bacterial adhesion and biofilm formation. In effect, an investigation of coated PET and polyamide with PEG of different molecular weights using a SiCl4 cold plasma treatment via the creation of C–Si–Clx functionalities permitting the covalent linkage of PEG macromolecules through a condensation reaction mechanism. Indeed, these coating showed significant inhibition of attachment and biofilm formation by Listeria monocytogenes and Salmonella enterica sv. Typhimurium compared to unmodified PET and polyamide (Dong et al. 2011). In addition, a research analyzed the coating of PEG-like compounds, 1,4,7,10-tetraoxacyclododecane ether and tri(ethylene glycol) dimethyl ether, onto stainless steel by a cold-plasma enhanced technique. The coatings were more hydrophilic and less rough than the uncoated stainless steel. Biological testing on a mixed culture of Staphylococcus epidermidis, Salmonella Typhimurium, and Pseudomonas fluorescens revealed a reduction in the bacterial adhesion and biofilm formation (Denes et al. 2001). In another study, the PEG-like compound, di(ethylene glycol) vinyl ether was deposited onto stainless steel surface via radiofrequency–plasma processes. These deposited films showed a stable chemistry and a more hydrophilic character and a decrease in roughness values in comparison with bare stainless steel. These new characteristics leaded to an effective anti-adhesive behavior of the coatings towards Listeria monocytogenes strains (Wang et al. 2003). Plasma treatments can be used as preliminary preparation for surface grafting. A research investigated the antifouling characteristics of grafted plasma-modified PET surfaces. In fact, two different gases, oxygen and helium, were employed to create superhydrophilic surfaces with various surface chemistries. Oxygen reactive gas used in plasma treatment increases the oxygen groups and enhances the hydrophilic character on the surface (Krstulović et al. 2006). Researchers demonstrated an antibiofilm activity of 3D printed polylactic acid petri dishes treated surfaces. Atmospheric pressure plasma was employed for the polymerization and deposition of acrylic acid. Plasma polymerization caused an increment of oxygen polar groups (C—O and O-C=O) producing a hydrophilic character of the coatings. This hydrophilic character played an essential role in Pseudomonas aeruginosa and Staphylococcus aureus biofilms reduction (Muro-Fraguas et al. 2020).

The employment of stainless steel

In food manufacturing and hospitals, cleaning and disinfection are paramount. The material and equipment selected upstream will affect the future care and disinfection methods. In those fields, metals specifically, can provide exceptional strength properties. However, strength is not the only aspect to consider. The most adequate material needs to be as inert and non-corrosive as possible.
Stainless steel is characterized by the addition of chromium at least 10.5 % of total composition. Chromium is very reactive to oxygen and immediately forms a strong barrier on its outer surface. This barrier is highly resistant and protects internal structures from additional corrosion [23]. Indeed, this stainless steel alloy is one on the most largely used material in biomedical and food fields. This metal is selected for its mechanical and chemical stability, biocompatibility, good corrosion resistance, low price and non-toxicity. It is mostly employed in medical sectors for orthopedic implants and prosthesis, cardiovascular valves and stents, and also for 3D printing of custom-made implants [24]. Moreover, in agri-food industries, stainless steel is selected since it does not affect the food’s colour or taste without contaminating it. It also provides amazing performance for maintaining food safety by being effortlessly and efficiently washed up and sterilized [25].
It is of importance to note that the major issue for researchers is to provide antibacterial and antiadhesive properties to implantable stainless steel. Moreover, it is difficult to elaborate coatings with specific mechanical properties and adequate antimicrobial/antiadhesive effect at the same time. That is why scientists tried to modify stainless steel surface via multiple strategies and elaborate coatings with the employment of several antimicrobial molecules.

Antimicrobial coatings on stainless steel incorporating biocides

Since ancient times, the most frequently used antibacterial heavy metal is silver. This metal species fights bacteria by disrupting enzymatic activities, disabling the membrane function, damaging the DNA and oxidative stress [26]. Moreover, silver was demonstrated to exhibit an excellent bacteriostatic and bactericidal effect towards several bacterial species [27]. Different configurations of silver were used to develop the films. Ions, metallic nanoparticles and silver halide nanoparticles were integrated into Layer by Layer (LbL) coatings on stainless steel and afterward released to kill bacteria. AgNPs has been extensively admitted to possess effective antimicrobial properties related to its oligodynamic action and multiple modes of its biocidal action [28]. Silver-based nanoparticle mixed with a cationic polymer poly(3,4-dihydroxy-L-phenylalanine)-co-poly(2-(methacryloxy)ethyl trimethylammonium chloride) (DOPA) permitted the adhesion enhancement of the LbL coating to stainless steel surface. The mixture formed an aqueous suspension with stable AgO and AgCl nanoparticles which was gathered with the polyanion poly(styrene sulfonate) (PSS) on stainless steel surface. Micelles were formed by the interaction between the positively charged DOPA and the negatively charged PSS. This LbL coating imparted an efficient antibacterial activity, related to the silver ions release from the film, against Escherichia coli strains [29,30]. Moreover, antimicrobial coatings were elaborated by electrodeposition of Ag on stainless steel from an AgNO3 aqueous solution. These films were designed for fracture repair implant and harmless to human osteoblasts preventing in vivo bacterial infection. The coatings were challenged with Pseudomonas aeruginosa and 13-fold bacterial reduction was observed after 24 h [31]. Otherwise, Cowan et al. [9] evaluated the antibacterial efficacy of stainless steel coupons coated with a zeolite matrix containing silver and zinc (AgION). These coatings showed an antimicrobial behavior against Gram-positive strains like Listeria monocytogenes and Staphylococcus aureus and on Gram-negative strains like Pseudomonas aeruginosa and Escherichia coli. In another study, antibacterial coatings based on chitosan and bioglass particles were developed on stainless steel using electrophoretic deposition (EPD). These coatings showed an efficient antibacterial activity against Staphylococcus aureus [32]. Moreover, stainless steel coatings based on AgNPs thin layers elaborated by reduction of Tollen’s reagent were developed using a formaldehyde-radiofrequency plasma functionalization. These coatings’ antimicrobial efficacy was tested and resulted in 5-log reduction in Listeria monocytogenes populations after 5 h of exposure [33].
Several studies concerning AgNPs toxicity reported that a safe range can be established for the use of AgNPs in designing antimicrobial coatings [28]. However, despite silver’s effective antibacterial activity, the toxic effect of AgNPs against the mammalian cells limited their use [34]. Moreover, Sung et al. [35] outlined that a constant exposure to AgNPs beyond 90 days caused inflammatory lesions that considerably weakened lung function of rats.

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Light-Activated Antimicrobials coated on stainless steel

Antimicrobial coatings can also be developed using light activated antimicrobial that are photo-activated compounds. Those species might be organic, aromatic like porphyrin, chlorophyll derivatives or inorganic oxides like titanium dioxide (TiO2). The antimicrobial activity become efficient when those compounds are exposed to light with a specific wavelength. The reaction gives reactive oxygen species that interact with the bacterial protective membranes and DNA and results in bacterial death [43,44]. These species are regarded as bacteriostatic rather than bactericidal [45]. Regarding stainless steel coated substrate with those photo-activated compounds, a study involved grafting a thin film of TiO2. This modified stainless steel showed an efficient antibacterial activity against Bacillus pumilus [11]. Moreover, the frequently touched surfaces play an important role in the spread of bacterial infections in hospitals. In a research, nanostructured TiO2 coatings on stainless steel surface were elaborated. They were made via chlorine chemistry and elaborated to kill bacteria under visible light, via an enhanced photocatalytic activity. These coatings showed greater than 3-log reduction in viable Escherichia coli after 4 hours visible light exposure [46]. However, some of these antimicrobial coatings can only be activated by UV light or by very high intensity light sources which may be dangerous to human health [47].

Cationic molecules coated on stainless steel

Cationic molecules have the ability to impart a bactericidal activity while released from an LbL film or when are immobilized on the surface. They can be bacteriostatic when employed at a lower rate than the minimum inhibitory concentration (MIC) and bactericidal when used at higher concentration than the MIC [48]. Bacteria will be killed once in contact with these positively charged molecules. The antimicrobial properties of the cationic functional groups stabilized on a surface persist more than the released ones [30]. In a research, a type of coating using a polycation has been generated to impart antifouling and antimicrobial properties to stainless steel surface. Chitosan, a versatile hydrophilic polysaccharide was used for its large antimicrobial spectrum. The stainless steel surface was grafted with chitosan via polymer brushes based on poly (2-hydroxyethyl methacrylate) (PHEMA). The coating was developed using LbL process, firstly, as an initiator, a layer of barnacle cement (BC) holding the alkyl bromide, an atom transfers radical polymerization (ATRP) of 2-hydroxyethyl methacrylate, was fixed to the surface. The hydroxyl groups of PHEMA were then converted to carboxyl groups for linking to chitosan. The coated surface reduced bacterial adhesion and presented an antibacterial efficacy against Escherichia coli [12].
The main positively charged structure used for its bactericide power are quaternary ammonium compounds (QACs) [49]. In a study, QACs were coated on silanized stainless steel substrates via alkylation of immobilized ethylene diamine using cold plasma techniques. The coated films showed bactericidal efficacy against Staphylococcus aureus and Klebsiella pneumonia [50].

Enzymes coated on stainless steel

Antimicrobial enzymes are ubiquitous in nature, participating meaningfully in the defense mechanisms of organisms against bacterial infection and fungi. They are wall-degrading enzymes that result in cell wall breakage and cell death [59]. They can be bacteriostatic and bactericidal depending on the concentration employed [60]. Antimicrobial enzymes are also employed for bactericidal film development. For example, stainless steel, pretreated with poly(ethylene imine) (PEI), was grafted with lysozyme and/or poly(ethylene glycol). It showed an antimicrobial activity against Listeria ivanovii and Micrococcus luteus [14]. Moreover, the efficiency of serine protease trypsin in preventing biofilm formation was reported [61]. The trypsin-grafted bioactive coating was developed on stainless steel showed an efficient antibacterial activity against Staphylococcus epidermidis biofilm [61]. Despite their effectiveness, the economic cost for enzymes extraction and purification before use is very expensive [62].

Table of contents :

CHAPTER: I Cold plasma surface treatments to prevent biofilm formation in food industries and medical sectors
Abstract
Introduction
Factors influencing the bacterial adhesion to a substrate
Cold plasma technologies
Approaches for cold plasma surface modification
Plasma physico-chemical modification to surface
Cold plasma applications
Cold plasma treatments of materials for preventing bacterial adhesion
Conclusion
References
CHAPTER: II Antimicrobial peptides coated stainless steel for fighting biofilms formation in food and medical fields
Abstract
Introduction
The employment of stainless steel
Antimicrobial coatings on stainless steel incorporating biocides
Silver nanoparticles coated on stainless steel
Essential oils coated on stainless steel
Light-Activated Antimicrobials coated on stainless steel
Cationic molecules coated on stainless steel
Antibiotics coated on stainless steel
Enzymes coated on stainless steel
Generalities on antimicrobial peptides
Stainless steel coatings incorporating antimicrobial peptides
Nisin qualification
Nisin antimicrobial properties and mechanism of action
Nisin antimicrobial spectrum and applications
Conclusion
CHAPTER: III Cold plasma assisted deposition of organosilicon coatings on stainless steel for Salmonella enterica serovar Enteritidis adhesion prevention
Abstract
Introduction
Materials and Methods
Materials, bacterial strains and culture condition
Plasma coatings Elaboration
Fourier transform infrared characterization
Water contact angle measurements
MATS: Microbial affinity to solvents
Bacterial suspension and adhesion test
Surface roughness and thickness analyses
Evaluation of coating adhesion to stainless steel
Scanning electron microscopy analyses
Statistical analysis
Results
Chemical properties of TMDS coatings
Anti-adhesive character of TMDS coatings
Topography of TMDS coatings
Discussion
Conclusion
References
CHAPTER: IV Nisin-based coatings for the prevention of biofilm formation: Surface characterization and antimicrobial assessments
ABSTRACT
Introduction
Material and methods
Approaches for coatings elaboration
Standardized SS slides preparation
Bacterial strain, culture conditions and suspension preparation
Protocols for coatings elaboration
 Stainless steel/polydopamine (SD) coatings
 Stainless steel/polydopamine/nisin (SDN) coatings
 Stainless steel/polydopamine/glutaraldehyde/nisin (SDGN) coatings
 Stainless steel/polydopamine/succinic acid /nisin (SDAN) coatings
Bacterial adhesion tests in NEC biofilm system
Antibacterial challenge test
Assessment of the bacterial viability with the Live/Dead backlight viability kit
Qualitative antibacterial assessment of treated surfaces
Water contact angle measurements
Surface roughness and thickness analyses
Ion polishing of coatings
Fourier Transform Infrared analysis
ToF-SIMS analysis
XPS analysis
Statistical analysis
Results
Antibacterial effect of nisin coated SS
Surface characterization of SS coated films
Discussion
Conclusion
References
CONCLUSIONS AND PERSPECTIVES
References

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