Atmospheric pressure nonthermal plasmas for bacterial biofilm prevention and eradicationa) Svetlana A. Ermolaeva, Elena V. Sysolyatina, and Alexander L. Gintsburg Citation: Biointerphases 10, 029404 (2015); doi: 10.1116/1.4914382 View online: http://dx.doi.org/10.1116/1.4914382 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/10/2?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Optical and structural properties of plasma-treated Cordyceps bassiana spores as studied by circular dichroism, absorption, and fluorescence spectroscopy J. Appl. Phys. 117, 023303 (2015); 10.1063/1.4905194 A study of oxidative stress induced by non-thermal plasma-activated water for bacterial damage Appl. Phys. Lett. 102, 203701 (2013); 10.1063/1.4807133 Atmospheric pressure resistive barrier air plasma jet induced bacterial inactivation in aqueous environment J. Appl. Phys. 113, 093302 (2013); 10.1063/1.4794333 Mass spectrometric study on inactivation mechanism of spore-forming bacteria by low-pressure surface-wave excited oxygen plasma Appl. Phys. Lett. 98, 191501 (2011); 10.1063/1.3588036 The hairline plasma: An intermittent negative dc-corona discharge at atmospheric pressure for plasma medical applications Appl. Phys. Lett. 96, 143701 (2010); 10.1063/1.3380811

Atmospheric pressure nonthermal plasmas for bacterial biofilm prevention and eradicationa) Svetlana A. Ermolaevab) Gamaleya Institute of Epidemiology and Microbiology, Gamaleya St. 18, Moscow 123098, Russia

Elena V. Sysolyatina Gamaleya Institute of Epidemiology and Microbiology, Gamaleya St. 18, Moscow 123098, Russia and Moscow Institute of Physics and Technology, Dolgoprudnyi, Moscow 141700, Russia

Alexander L. Gintsburg Gamaleya Institute of Epidemiology and Microbiology, Gamaleya St. 18, Moscow 123098, Russia

(Received 11 December 2014; accepted 25 February 2015; published 13 April 2015) Biofilms are three-dimensional structures formed by surface-attached microorganisms and their extracellular products. Biofilms formed by pathogenic microorganisms play an important role in human diseases. Higher resistance to antimicrobial agents and changes in microbial physiology make treating biofilm infections very complex. Atmospheric pressure nonthermal plasmas (NTPs) are a novel and powerful tool for antimicrobial treatment. The microbicidal activity of NTPs has an unspecific character due to the synergetic actions of bioactive components of the plasma torch, including charged particles, reactive species, and UV radiation. This review focuses on specific traits of biofilms, their role in human diseases, and those effects of NTP that are helpful for treating biofilm infections. The authors discuss NTP-based strategies for biofilm control, such as surface modifications to prevent bacterial adhesion, killing bacteria in biofilms, and biofilm destruction with NTPs. The unspecific character of microbicidal activity, proven polymer modification and destruction abilities, low toxicity for human tissues and absence of long-living toxic compounds C 2015 American Vacuum make NTPs a very promising tool for biofilm prevention and control. V Society. [http://dx.doi.org/10.1116/1.4914382]

I. INTRODUCTION Infectious disease remains an important problem in modern medicine. A wide spread of polyresistant strains and formation of biofilms by the most ubiquitous pathogens considerably lessen the effectiveness of antibiotic treatment. The National Institutes of Health (United States) estimates that 80% of all infections are biofilm-related.1 Biofilms formed by pathogenic bacteria are responsible for biomaterial-related infections, nonhealing chronic wounds, dental diseases, endocarditis, etc. Meanwhile, the cost of chronic wound healing exceeds $10 billion annually in the United States alone and constitutes over half of the total cost for all skin diseases.2 Biofilm-related implant infections are the most common cause for revision surgeries in total-knee arthroplasties (25%), the third most common cause in hip arthroplasties (15%), and the most common reason for knee arthroplasty and hip arthroplasty removals (79% and 74%, respectively), costing about US$70 000 per episode.3 Treatment of biofilms associated with periodontal diseases costs tens of billions of dollars every year. Biofilm-related diseases bring and urgent need to develop methods of biofilm inactivation and eradication. Meanwhile, Costerton et al.4 emphasized that the therapeutic strategies that have served very well in the eradication of acute epidemic infectious diseases have not yielded favorable a)

Published without proof corrections from the authors. Electronic mail: [email protected]

b)

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outcomes when applied to the biofilm diseases. Higher resistance to antimicrobial agents, changes in microbial physiology, and toxic compounds accumulated in the biofilm matrix make treatment of biofilm infections difficult and necessitate the development of new therapeutic approaches. This review focuses on biofilm physicochemical and biological properties, the impacts of biofilms on human diseases, and current and potential use of nonthermal plasmas (NTPs) for biofilm inactivation and eradication. Strategies of biofilm prevention and removal that can be developed on the basis of NTP applications are discussed. II. STRUCTURE AND PROPERTIES OF BIOFILMS Biofilms are defined as three-dimensional structures formed by assemblages of microorganisms typically attached to a surface and their associated extracellular products.5 The surface-attached configuration and biofilm formation are preferred for microorganisms in natural habitats. In most habitats, including soil, water reservoirs, and extreme environments such as acid mine drainage areas, hot springs, and ice deserts, microbial biofilms are a predominant and sometimes unique form of life. Normal human microflora inhabiting mucosal surfaces of the gut, pharynx, etc., exist in the biofilm mode. Generally speaking, biofilms represent a dominant form of microbial existence under the most divergent conditions. However, when biofilms are formed by pathogenic species, they cause serious problems for human medicine. Biofilm-infected medical devices, catheters,

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C 2015 American Vacuum Society V

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cannulas, surfaces, and sewer systems in hospitals are the most common cause of nosocomial infections. In the course of infection of a human body, biofilms delay apparent clinical recovery, defending pathogens from both immune defense mechanisms and medications. Improved resistance of biofilms to standard antimicrobial treatments has generated an urgent need for novel approaches. A. Biofilm formation

Biofilm formation includes several stages. The initial adhesion is followed by formation of microcolonies by the first colonizers [Fig. 1(a)]. The microcolonies expand and merge upon biofilm maturation. Still, the structural features of microcolonies are often evident even in mature biofilms [Fig. 1(b)]. Mature biofilms represent cell aggregates interspersed within a polymeric matrix. The process of bacterial exit from mature biofilms into a planktonic state is known as biofilm dispersal. The extracellular matrix produced by the bacterium plays the most important role in biofilm maturation and maintenance. Exopolysaccharide, such as poly-N-acetylglucosamine, is the most abundant component of the matrix and plays an important role in virulence.7,8 Other components are proteins and extracellular DNA generated by bacterial lysis.1 B. Factors that control bacterial adhesion

Biofilm formation begins with bacterial attachment to a surface. This step is determined by intrinsic bacterial features and by properties of the surface. Both specific molecular structures and unspecific physicochemical interactions play an important role in adhesion. Such bacterial surface structures as flagella, pili, and adhesins are required for initial interaction of a motile bacterium with the colonizing surface (reviewed in Ref. 9). Thermodynamic analyses of the bacterial cell and the surface interactions led to the conclusion that bacterial surface adhesion is generally dominated by short-range attractive Lewis acid–base interaction forces, in combination with long-range, weaker Lifshitz–van der Waals forces.10 Physicochemical properties of the surface exert a strong influence on the attachment process. Surface roughness,

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positive charge and hydrophobicity are the three important factors that favor initial bacterial attachment.9 Fletcher and Loeb11 performed a comprehensive study of the attachment of a marine Pseudomonas spp. to a variety of surfaces that revealed dependence on the surface charge and degree of hydrophobicity. Hydrophobic plastics with little or no surface charge [Teflon, polyethylene, polystyrene, and poly(ethylene terephthalate)] had the highest adhesion efficiency, followed by hydrophilic metals with positive or neutral surface charges. Adhesion efficiency was further decreased for hydrophilic, negatively charged nonmetallic surfaces (glass and oxidized plastics). Separate adhesion mechanisms for hydrophobic and hydrophilic surfaces were suggested by Paul and Jeffrey12 in their study of the marine bacterium Aeromonas proteolytica. The inherent surface charge of a microbial cell considerably affects adhesion. Most bacteria are negatively charged, favoring attachment to positively charged surfaces and improved surface interactions due to increased ionic strength.13 However, a positive cell surface charge occasionally may occur among bacteria or fungi, favoring attachment to negatively charged surfaces may.14,15 The distinct mechanisms responsible for hydrophilic and hydrophobic bacterium/surface interactions correlate to a range of surface wettability optimal parameters. Rosenman and Aronov16 performed a comparative study of bacterial adhesion on the surfaces with the contact angle (H) varied within a range of 20 (hydrophilic surface) to 100 (hydrophobic surface), which demonstrated an opposed behavior of different bacterial species. While Escherichia coli adhered on hydrophilic surfaces (H  20 –40 ), another Gram-negative bacterium Pseudomonas putida adhered mainly on the hydrophobic surfaces (H  80 –100 ). Adherence was prevented for all tested bacteria when the contact angle was near 60 . The ability of the surface to absorb organic molecules, forming so-called conditioning film, plays an important role in bacterial attachment and biofilm formation.17 The accumulation of proteins, polysaccharides, and other molecules on the surface provides a metabolically favorable environment for microbial cells and serves as nutritional cues to trigger biofilm formation.

FIG. 1. 3D image of young (a) and mature (b) P. aeruginosa biofilms made with confocal laser scanning microscopy. (a) P. aeruginosa adhesion to glass; the initial stage of P. aeruginosa biofilm formation (6 h). (b) The microcolony structures maintained in the mature biofilm (36 h) are enlarged. [E. Sysolyatina, N. Kaminskaya, and S. Ermolaeva (unpublished)] (Ref. 6). Biointerphases, Vol. 10, No. 2, June 2015

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C. Biofilm structure

The structure of a biofilm considerably depends on the microorganisms forming it and the environmental parameters. For instance, Campanac18 found that biofilms formed by the Gram-positive bacterium Staphylococcus aureus are noticeably “poorer” than biofilms formed by Gram-negative bacteria Pseudomonas aeruginosa and include less proteins and polysaccharides under the same conditions. Besides cell and surface properties, the structure and organization of microbial biofilms are largely determined by available nutrients and fluid dynamic forces.5,19,20 For example, under high shear stresses, such as on the surface of teeth during chewing, the biofilm known as a dental plaque is typically stratified and compacted.5 Hydrodynamic conditions that control the two interlinked parameters of mass transfer and drag significantly influence processes of biofilm development.20 Biofilms grown under laminar conditions usually consist of roughly circular cell clusters separated by interstitial voids, while the turbulent-flow biofilms consist of patches of ripples and elongated “streamers.” Elaboration of the 3D structures of living biofilms revealed considerable heterogeneity in density of biofilm mass, including the presence of pillarlike and mushroomlike structures (Fig. 2).16 Water-filled channels are interspersed among biofilm strata. It has been suggested that the channels supply nutrient circulation within biofilms. Water channels were shown to be involved in passing hydrophilic antibiotics such as b-lactams through the entire thickness of the biofilm,22 thus reducing penetration of hydrophobic and charged substances. The most abundant component of the polymeric matrix covering microbial cells is negatively charged exopolysacharide (EPS). EPS has been shown to bind positively charged antibiotics, such as aminoglycosides, preventing their effects on bacteria. An increase in the molecule hydrophobicity results in a progressive loss of bactericidal efficiency as well.18 In addition to the preservation effect of the matrix, other welldocumented factors are involved in resistance of bacteria to antibiotics in biofilms. These factors are nutrient limitation,

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slow growth, adaptive stress responses, and formation of so-called persister cells.23–25 All these factors result in a slowing of metabolic processes, and thus, weaken the impact of antibiotics on bacterial viability. Those antimicrobials that affect metabolically inert resting cells are effective against microorganisms in biofilms, e.g., antimicrobial cationic peptides that target the microbial membrane.26 D. Biofilms as multispecies community

Natural biofilms represent complex structured communities that include multiple species. In the course of initial microcolony growth and biofilm maturation, bacteria belonging to distinct species physically interact with each other to form a common structure. The process of interspecies interactions in the course of biofilm formation is known as a coaggregation. Coaggregation was first described27 for dental plaque bacteria. Biochemical properties of each species are utilized for the welfare of others to compensate for their inabilities. This feature is particularly important for human pathogens as interspecies interactions between pathogens in mixed biofilms can enhance the production of virulence factors and alter the course of polymicrobial infections. When human bacterial pathogen P. aeruginosa is grown in mixed biofilm with Candida albicans, a polymorphic fungus that is among the normal microflora in the human gut, the production of pyoverdine, rhamnolipids, and pyocyanin is increased.28 These factors are major contributors to the ability of this bacterium to cause disease. Mixed biofilms are common in lung infections. P. aeruginosa, which is a major bacterial species of cystic fibrosis lung microbial communities, facilitates microcolony formation29 for another important lung pathogen, S. aureus. III. ROLE OF BIOFILMS IN HUMAN DISEASE A. Dental plaque biofilms

Oral biofilms—better known as dental plaques—include more than a hundred bacterial species and represent the best studied examples of interspecies interactions in biofilm formation. Biofilm formation on tooth surfaces usually depends

FIG. 2. 3D view of mature biofilms formed by foodborne pathogens L. monocytogenes, EGDe, and E. coli O157:H7 formed on vegetable tissue. The pillarlike structures are clearly visible. Image created using scanning electron microscopy (magnification 12 000 and 6000 for (a) and (b), respectively, unpublished, courtesy of V. I. Pushkareva) (Ref. 21). Biointerphases, Vol. 10, No. 2, June 2015

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on formation of a conditioning film (see above) that covers enamel and abridges subsequent cell-to-surface attachment of the primary colonizers.5,25 Initial biofilm formation was shown30 to be a result of adhesion and coaggregation between Streptococcus spp. and Actinomyces spp. Subsequent events are dependent on the ability of the next colonizers to interact efficiently with the first inhabitants of the biolfilm. For example, Actinobacillus actinomycetemcomitans, which is the latter colonizer of dental plaques, is unable to adhere to the film formed by S. gordonii,31 which is one of the most common first colonizers. However, A. actinomycetemcomitans can adhere to the film formed by the Gram-negative bacterium Fusobacterium nucleatum, which can aggregate with both species. Therefore, F. nucleatum serves as a bridge between S. gordonii and A. actinomycetemcomitans.32 The latter colonizers such as Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticolat are considered true periodontal pathogens. These results are particularly interesting for managing dental plaque, as they suggest different bacterial targets for plaque prevention and destruction. The specificity of interactions between different waves of colonizers depends on the specific interactions of bacterial surface structures. Oral bacteria were shown to have multiple adhesins, many of which are lipoproteins belonging to the lipoprotein receptor antigen I (LraI) family of lipoproteins and have been shown to be involved not only in coaggregation, but also in binding components of the salivary pellicle.33 Adhesins recognize glucanlike molecules on the surfaces of bacteria with which they coaggregate. They also recognize linear cell wall polysaccharides that contain characteristic repeating units, including N-acetylglucosamine.32,33 Coaggregation in the course of dental plaque formation requires specific amino-acid modification of lipoteichoic acids and calcium binding molecules from Gram-positive bacteria.34 The complex character of interactions between members of dental plaque biofilms suggests that differences in applied methods and compositions of antimicrobials might considerably influence the outcome of the plaque treatment. The importance of dental plaques is related to their role as a primary cause of oral diseases including caries, gingivitis, and periodontitis.35 Cariogenic microorganisms produce a number of organic acids, which cause a decrease in pH and result in demineralization of enamel hydroxyapatite (HA) crystals and proteolytic breakdown of the structure of tooth hard tissues. Streptococcus mutans, other streptococci of the nonmutans streptococci group, Actinomyces and Lactobacillus play a key role in this process. Metabolic changes in biofilms determine the alternating processes of decrease and increase of biofilm pH followed by the physical and chemical processes of demineralization and remineralization that are characteristic of caries.35,36 Mechanical hygiene (brushing and flossing) coupled with use of antimicrobials such as triclosan and triclosan/copolymer is a common way to control dental plaque in oral care. However, the increasing employment of triclosan and other antimicrobials in a range of consumer products has been severely criticized Biointerphases, Vol. 10, No. 2, June 2015

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for the lack of demonstrated benefits for hygiene and a risk of compromising their efficacy by wide-scale usage.37 Development of new approaches that both prevent plaque development and are potent enough to affect mature biofilms is an important challenge in current periodontology. B. Wound biofilms

Wound microflora and biofilms are less studied in comparison to dental plaques not only due to the difficulties in modeling, but mainly because of the presence of multiple, often anaerobic species that are difficult to cultivate, inhabiting the wound surface and contributing to the community survival.38 Skin and mucosal surfaces such as those of the oral cavity and the gut are the primary sources of microorganisms colonizing the wounds, which can be further colonized by incomers from the environment. The emergence of signs of infection depends on the composition of contaminating microflora as well as patient health and wound parameters.39 Infection of wounds contributes to chronic nonhealing wounds, chronic inflammation and results in the development of infection-related complications.40 P. aeruginosa, S. aureus, and other Staphylococci spp. are prevalent woundassociated pathogens.41,42 Among 13 wounds investigated in a study by Price et al., as many as 58 bacterial families were identified using molecular methods,43 as well as 91 bacterial genera, most of which are nonculturable. However, the impact of these multiple species on the development and persistence of infection in wounds has yet to be studied. In recent years, there have been multiple studies44–46 investigating the important role of biofilms in pathogenesis of chronic wounds. The preferable association of biofilms with chronic but not acute wounds has been demonstrated by James et al.,46 who detected the presence of biofilms in up to 60% of chronic wounds while only 6% of acute wound specimens were characterized as containing biofilms. This role also was suggested for biofilms formed by P. aeruginosa and other pathogens in chronization of venous leg ulcers, pressure ulcers, and diabetic foot ulcers. The biofilms provide an increased tolerance of pathogens to various antimicrobial measures and treatments, and protection from the phagocytic activity of polymorphonuclear neutrophils. This tolerance results in inefficient eradication of infecting agents and, therefore, inability to avoid activity of bacterial toxins and hyperinflammation.47,48 The detrimental effect of biofilms on wound healing makes mechanical removal of debris and biofilms critical in chronic wound therapy. Other potential strategies to combat wound biofilms include: prevention of bacterial attachment and further biofilm formation; disruption of the biofilm to allow penetration of topical antimicrobial agents; interference with bacterial signaling systems responsible for biofilm development; and enhancement of bacterial dispersion from biofilms to a more easily destroyed planktonic state.47 Though the methods for implementing suggested steps are still insufficient, some studies have made promising strides.

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A negative-pressure wound therapy with irrigation or instillation was demonstrated to lower the bacterial burden in chronic wounds and prevent biofilm formation.49 Application of NTP in conjunction with microbicidal and immunostimulatory topical solutions seems to be another multimodal strategy to combat chronic wounds. Redox imbalance along with biofilms was shown to contribute significantly to chronicity.50 However, because NTP is active against biofilms, it can change a redox balance affecting oxidative processes in the wound, thus encouraging wound healing. C. Biofilms associated with medical devices

Biofilms associated with medical equipment and devices are among the most important causes of nosocomial infections.51,52 A significant proportion of device-related infections are linked to occupation of medical implants by ubiquitously spread pathogens such as Staphylococcus spp. or P. aeruginosa. Biofilm-associated infections were registered for almost all types of implants including venous catheters, coronary stents and heart valves, ventricular assist devices, arthroprostheses, intraocular lenses, dental implants, etc. The importance of bacterial adhesion as an initial critical step in biofilm formation was discussed in previous sections. Here, we would like to emphasize the role of surface properties in implant colonization. Several recent conferences have recommended that researchers focus on the development of effective antibacterial surfaces53 that would prevent bacterial adhesion and biofilm formation. Surface modifications as a way of antibacterial treatments for biofilm prevention are especially important for treatment of implants assigned for prolonged (lifelong) operation. Implants must possess both structural and surface compatibility with the host tissues, which puts certain restrictions on materials used for their manufacturing. Hence, materials that are intended for fabricating orthopedic implants must bear high levels of mechanical stress. Metals (Ti-6Al-4V, Co-Cr-Mo and stainless steel), polymers [poly(methyl methacrylate) and ultrahigh-molecular-weight polyethylene], and ceramics (alumina, zirconia, and hydroxyapatite) are the three classes of most commonly used materials for orthopedic implants.54,55 When orthopedic implant materials were compared by bacterial attraction, metals were the most easily colonized by Staphylococci.54 This result is consistent with the attraction of negatively charged bacteria to hydrophilic metal surfaces with a positive or neutral surface charge (as discussed in previous sections). The ideal implant surface should allow human cell adhesion while preventing bacterial adherence. To optimize composite hydroxyapatite ceramics, such parameters as surface wettability and structure have been comprehensively studied.15,56–59 Several studies demonstrated56,57,59 that a maximally hydrophilic surface with contact angle H  0 was optimal for human osteoblasts adhesion. While improving human cell adhesion, the superhydrophilicity seems to prevent adhesion of Gram-positive bacteria,16,57 including S. aureus, which is one of the most Biointerphases, Vol. 10, No. 2, June 2015

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significant pathogens contaminating implants. Superhydrophobicity as well as super-hydrophilicity prevents bacterial attachment.58 Such characteristics as nanoscale surface roughness can be useful to decrease bacterial adhesion and biofilm development. When microscale roughness is considered, bacterial adhesion is facilitated by the increased surface area while surface irregularities might provide additional protection for adhered bacteria.60 However, exceptionally smooth materials experience bacterial attachment via physical forces such as van der Waals interactions and by providing a large number of molecular contact points.61–63 Moreover, Ivanova et al.62 observed an increased level of secretion of extracellular polymeric substances involved in biofilm formation on nanoscale smooth surfaces. Certain nanometer-sized surface topographies were shown to prevent bacterial adhesion while improving osteoblast attachment, proliferation, and differentiation, thus promoting bone tissue formation.62,63 IV. ATMOSPHERIC PRESSURE NONTHERMAL PLASMA APPLICATIONS FOR BIOFILM CONTROL Atmospheric pressure NTPs that are used in medical applications are close to ambient temperature. NTPs affect biological objects via a synergetic action of bioactive components such as charged and metastable particles, neutral active species, and UV photons.64,65 For the last 5 years, many investigations have been devoted to different aspects of biofilm control with NTPs. For the purpose of this review, these works can be divided into three categories as follows: (1) surface modifications by low-pressure and atmospheric pressure plasmas to prevent bacterial attachment and/or surface colonization; (2) bacterial killing within biofilms with NTPs; and (3) biofilm destruction with NTPs that suggests a decrease in the biofilm biomass, including cells and matrix. A. Surface modifications by low-pressure and atmospheric pressure plasmas to prevent bacterial attachment and/or surface colonization

Applications of nonthermal plasmas for surface modifications have a long history. In the past years, a number of comprehensive reviews have appeared, describing biocompatible modifications of implant and other medical devices by using different plasma-based techniques.66 Here, we bring our attention to the plasma-based surface modifications shown to prevent bacterial attachment and/or biofilm formation. There are two main approaches for surface modifications to reduce biofilm-associated infections. The first is incorporating microbicidal agents61,66,67 such as antibiotics, silver (Ag) nanoparticles, nitrofurazone, chlorhexidine, antibacterial peptides, etc., which kill attached bacterial cells and thus stop surface colonization. The second is surface modification to prevent bacterial adhesion16,63,68 by changing such important characteristics as surface chemistry, wettability, size, and porous distribution and roughness. Titanium (Ti) coating with HA via a plasma spray deposition is among the most frequent commercially available

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implant modifications in orthopedics and dentistry. Ti possesses superior specific strength, light weight, and corrosion resistance, while HA is a chemical analog to the inorganic phase of bone. HA films considerably improve cell adherence and bond tissue formation.69 Still, the microscale topography of the plasma-spray deposited HA film was shown to increase bacterial adherence,57 and it was suggested to be a cause of an observed increase in total hip revision surgeries. Studies on oral biofilms have demonstrated that the sequence of appearance of various microbial morphotypes is similar for enamel and Ti/HA dental implants.70 To prevent bacterial colonization, various bactericidal doping has been suggested. Silver (Ag) and silver oxide (Ag2O) with HA coating are widely studied due to the well-established bactericidal properties of Agþ.66,67 A number of other Ti coatings with antibacterial properties also have been proposed in recent years. Yoshinari et al.71 studied plasma-based Ti modifications by ion implantation and demonstrated the repressive effects of an alumina coating with formation of Al2O3, Agþ, and Znþ ions on adhesion of the periodontic-related bacterium P. gingivalis. In contrast, Ti nitrition did not influence attachment by either P. gingivalis or multispecies communities, while implantation of Ca ions increased bacterial adhesion.71 Lee et al.68 showed that hydrogenated Cu-incorporated diamondlike carbon (a-C:H/Cu) films, prepared using a radiofrequency plasma magnetron sputtering system, exhibited high hydrophobic surface features and a nanoscale structure that did not influence cell adhesion or proliferation behaviors, but still possessed excellent antibacterial properties. Mahfoudh et al.72 demonstrated that using dry-ozone exposure on polymeric surfaces to impart a microbicidal property is effective against sporulated bacteria. Another plasma-based approach for surface modifications is activation of chemically inert materials like silicone to provide binding sites for polymeric coatings, thus preventing bacterial adhesion. Hook et al.63 performed a highthroughput study using polymer microarrays that revealed important surface chemistry parameters that give polymers antiadhesion properties. The study also demonstrated the role of combining aromatic and aliphatic carbon groups with the ester group and weakly amphiphilic structures to yield antibacterial polymer properties. Prevention of bacterial adhesion appears to be an essential characteristic of future biocompatible materials. B. Microbicidal plasma action on microorganisms in biofilms

Microbicidal properties of nonthermal plasmas have been intensively studied on a wide range of microorganisms. Multiple factors acting in synergy are involved in microbicidal plasma effects and provide its almost unspecific activity toward bacteria and fungi. Still, the relative efficiency of bacterial killing significantly varied depending on both the microbial species and/or strain and the plasma composition. In general, fungi and bacterial spores are more resistant to NTP than vegetative bacterial cells.73–76 When Biointerphases, Vol. 10, No. 2, June 2015

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nonspore-forming Gram-positive and Gram-negative bacteria were compared, the results were controversial. In some cases, no significant differences were detected,74,76 while other researchers found that Gram-positive bacteria were more resistant than Gram-negative bacteria.75 Variations in the plasma devices and feeding gases used and, therefore, in plasma compositions were thought to be responsible for relative and absolute effectiveness of bacterial killing. In a previous study, we found77 that the relative resistances of Gram-positive S. aureus and Gram-negative P. aeruginosa were different when strains were tested with three different plasma sources in the same study. P. aeruginosa was more resistant to the corona discharge than S. aureus. In regard to the air plasma afterglow produced by the ferroelectric generator, S. aureus demonstrated similar or slightly higher resistance than P. aeruginosa to the microwave argon plasma. The time intervals required to kill 105 CFUs using corona discharge, a microwave plasma source and a ferroelectric generator were considerably different, at 10–120, 300, and 25 min, respectively. A discussion of mechanisms responsible for bacterial inactivation is beyond the scope of this review. It was presumed that charged particles and electromagnetic fields present in the direct plasma are responsible for observed distinctions in effectiveness of bacterial killing. Bacterial resistance depends on the structure of a cell wall as well as species- and strain-specific variability in stressresponse mechanisms.76–79 The microbicidal plasma effect on microorganisms in biofilms depends on intrinsic bacterial susceptibility and NTP characteristics in the same manner as it was shown for individual microbial cells. Table I gives several examples of effectiveness of microorganism killing in biofilms. Matthes et al.83 reported a 7.11- and 3.38 log decrease in the amount of living bacteria in P. aeruginosa and S. epidermidis biofilms, respectively, which were treated with the structured electrode planar SBD surface barrier discharge for 600 s. However, it takes only 240 s to kill 109 S. epidermidis in the biofilm treated with the plasma jet operated on mixed argon and air (9/1).85 One of the was described by Gorynia92 described one of the lowest sensitivities to plasma jet irradiation, for Streptococcus sanguinis biofilms, which experienced less than 1-log decline in CFUs after 180 s of treatment. When sensitivity of agar-plated planktonic cells to microwave argon plasma was compared, it was found that Streptococcus spp. were among the most resistant species to NTP.95,96 In addition, strain-specific differences in resistance to killing with NTP were observed for planktonic cells of microorganisms in biofilms. Sun et al.94 reported differences in survivor kinetics curves for biofilms formed by distinct C. albicans strains. The available set of plasma active particles considerably affects the antimicrobial efficiency. The plasma afterglow seems to be less effective for biofilm inactivation, which is consistent with a low bactericidal activity for nondirect plasmas. For instance, Salamitou et al.86 reported a decline in the amount of living bacteria in E. coli biofilms by a factor of 40 after a 40-min treatment with N2/O2 plasma afterglow.

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TABLE I. Nonthermal plasma activity against biofilms.

Microorganism

Plasma source

Feeding gas

Bactericidal effect (log 10 CFU reduction)

Time to reach the effect

Reference

P. aeruginosa

Plasma jet (13.56 MHz, 100 W) Plasma jet (13.56 MHz, 100 W) Plasma jet (20 kHz, 6 kV) Structured electrode planar SBD surface barrier discharge (SBD; 20 kHz, 13 kV) Floating-electrode dielectricbarrier discharge (500 Hz, 15 kV) Structured electrode planar SBD surface barrier discharge (SBD; 20 kHz, 13 kV) Plasma jet (100 kHz) Postdischarge plasma afterglow (28 kHz; 10 kV) Plasma jet (10 kHz, 8 kV) Plasma jet Plasma brush Plasma jet (2.45 GHz) Plasma brush Plasma jet (10 kHz; 10 kV) Plasma jet (1.8 MHz, 170 V) Plasma jet (2.45 GHz) Surface microdischarge (1 kHz, 9 kV) Plasma microjet

He/N2 20.4/0.135 He/N2 20.4/0.15 He/O2 99.5/0.5 Air

7 7.12 4 7.11

180 s 300 s 240 s 600 s

80 81 82 83

Air

7

60 s

84

Air

3.38

600 s

83

Ar/air 9/1 N2/O2 80/20

9 1.6

240 s 40 min

85 86

Ar He/N2 20.4/0.305 Ar /O2 He/O2/N2 2/1.2/1.5 Ar/O2 He/O2 2/0.04 Ar He/O2/N2 2/1.2/1.5 Air

>2 >2 7.23 4 6.84 7 0.6 5 5

300 s 300 s 13 s 18 s 13 s 20 min 180 s 12 s 7 min

87 88 89 90 89 91 92 90 93

He/O2 98/2

>2

60 s

94

S. aureus Staphylococcus epidermidis

E. coli P. gingivalis Chromobacterium violaceum S. mutans Lactobacillus acidophilus Neisseria gonorrhoeae Streptococcus sanguinis C. albicans

Bacteria in biofilms demonstrated higher resistance to plasma treatment when compared with planktonic bacterial cells of the same strain.88,91,95 These results suggested a higher level of resistance to NTP and involvement of parameters other than those influencing planktonic cell resistance. Besides the specific traits of microorganisms and the plasma bioactive component composition, biofilm thickness and structure appear to contribute to NTP resistance. In a previous study, we suggested that the bactericidal plasma effect depends on the thickness of a biofilm, in response to significant scattering observed in the data obtained from genetically modified B. cenocepacia strain Bc 46, an unstable biofilm producer.95 A microscopic study of bacterial viability on various layers of P. aeruginosa biofilms revealed higher concentrations of living bacteria in deeper layers, while dead bacteria prevailed at upper layers of plasmatreated biofilms (Fig. 3).82,95 The debris formed by dead upper bacteria and the matrix itself may contribute to the survival of bacteria in deeper locations.82,83,88 The specific changes in physiology that take place in the course of biofilm development also may affect resistance of microorganisms. However, the role of microbial physiology in resistance to NTP has yet to be described. C. Biofilm destruction with NTP

The role of the biofilm matrix in human diseases is often underestimated. Meanwhile, the matrix not only protects embedded microorganisms from antimicrobials and other Biointerphases, Vol. 10, No. 2, June 2015

unfavorable environmental conditions, but also contributes pathogenesis in other ways. Particularly, components of the matrix enhance tight adherence and stimulate bacterial accumulation on the surface via improving mechanical integrity. It also may act as a reserve source of energy helping to concentrate nutrients and ions in the microenvironment. Finally, it may accumulate toxins and other virulence factors produced by microorganisms.4,5,97 Therefore, methods that kill bacteria and also destroy the matrix are highly relevant. Plasma reactive particles produce a general mechanical effect on the polymeric surface referred to as “etching.”98–102 Etching with low-pressure CF4-O2 plasmas is one of the key processes used in microelectronic fabrication. Generation of volatile by-products that are subsequently desorbed from the surface as a result of reactions of highly reactive gas radicals with organic materials was shown to be the main mechanism of etching at low pressures. Although the etching effect of atmospheric pressure plasma is less studied, its usefulness for biofilm eradication has been demonstrated by recent studies. Fricke et al.103 found that etching rates of a high-frequency atmospheric pressure plasma jet fed with the Ar/O2 mixture varied between 50 and 300 nm s1 when applied to carbonbased aliphatic and aromatic polymers. Such rates are highly suitable for removing biofilms with thicknesses in the range of tens of micrometers. Indeed, the same investigators104 reported the total elimination of 7-day old C. albicans biofilms with thicknesses of 10–20 lm by etching for 300 s at rates of 33–67 nm/s. Rupf et al.105 reported total removal of 91-lm thick dental plaques formed in-situ on titan disks, by a

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FIG. 3. Differentially labeled P. aeruginosa in biofilms. Live bacteria are green, and dead bacteria are red. The distance from the glass surface is shown (in lm) in the upper left corner of each section. The left panel [(a)–(c)] shows control samples and the right panel [(d)–(f)] shows biofilms. Bacteria were treated with microwave argon plasma for 5 min. The prevalence of dead bacteria in the upper layers of the plasma-treated biofilm can be seen (Ref. 95).

sequential application of repeated treatments with a microwave-driven He plasma jet alternating with an air/water spray to remove detached debris. Vandervoort and BrellesMarino80 studied destruction of P. aeruginosa PAO1 biofilms with He/N2 (20.4/0.135) plasma generated by the RF plasma jet. Near-complete destruction of the matrix was achieved after 30 min of treatment. However, even after matrix destruction, bacterial cells were still found on the glass surface. In contrast, Traba et al.106 reported total removal of S. aureus bacteria, or at least nucleic acids of the bacterial Biointerphases, Vol. 10, No. 2, June 2015

origin, while polymer residues remained even after a 60-min application of O2 NTP. Diverse methods used for visualization of biofilm remnants and differential NTP properties may be responsible for discrepancies in the results of independent studies. The mechanisms of plasma etching for different atmospheric pressures are not yet fully understood. The works of Fricke et al.103,104 highlighted the important role of oxidation processes that appear to be a main driving cause of bond-breakage in organic polymers treated with NTP.

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Fragmentation of polymers can result in the formation of volatile fragments that may easily be removed from the surface, e.g., with water spray, as was shown by Rupf et al.105 Indeed, using atomic force microscopy, Vandervoort and Brelles-Marino80 discovered that plasma treatment induced a decline in the adhesiveness of P. aeruginosa biofilms, presumably caused by destruction and removal of the matrix. Further studies of physicochemical interactions between atmospheric-pressure plasma and organic matters, and biofilms themselves, will provide clearer understanding of biofilm etching and destruction mechanisms. It is important to note that biofilm fragments formed during the process of biofilm destruction could contribute to the spread of infection.4 Even in the absence of living bacteria, toxin-containing fragments of the matrix are undesirable elements that can provoke toxic effects in remote parts of the organism. Awareness of the sequential character of the NTP destructive action that begins from upper layers of the biofilm may be helpful to escape these complications in the course of biofilm etching. V. CLINICAL RELEVANCE OF ANTIBIOFILM NTP ACTIVITIES Antimicrobial and antibiofilm properties of NTP are important for many applications in medical practice, and especially in dentistry and chronic wound healing. Developing appropriate technologies for modern medicine poses new challenges that have not been previously identified. Recent achievements in implantology have raised the issue of peri-implant diseases (peri-implantitis) in dentistry. Biofilms formed on the microstructure Ti implants are among the main causes of the peri-implant parodont disease.107 The demonstrated effectiveness of NTP-assisted biofilm removal from dental implants opens new perspectives for the prevention and therapy of these complications.105 Since the role of root canal biofilms in a wide spread of peridodontic diseases was confirmed,108,109 the development of methods for effective root canal sterilization is the top priority in dental research. Radiofrequency plasma jets were demonstrated to be a promising tool for biofilm inactivation within a root canal. Depending on experimental settings, RF NTP was shown to kill between 90% (Ref. 110) and 100% (Ref. 85) of bacteria in biofilms within the root canal. Application of plasma-treated liquid to infected root models showed a sterilization rate of 97.4%.96 The efficiency of NTP in root canal sterilization is comparable to traditional methods and a combination of approaches may be useful to achieve total biofilm elimination. Coban et al.111 demonstrated an additive antimicrobial effect between argon plasma and chemical agents such as 0.1% chlorhexidine, 0.1% octenidine, 0.6% hypochloride, or 1.5% H2O2 that increases efficiency of multispecies subgingival biofilm inactivation. Enhancement of microbicidal effects of traditional medicine is yet another fast-developing area of plasma medical research. Poor et al.112 reported that NTP treatment added strong antimicrobial properties to the alginate gel that is Biointerphases, Vol. 10, No. 2, June 2015

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already in clinical use as a topical wound treatment, but that alone has no meaningful antimicrobial properties. The treated gel had the potential to inactivate up to 109 CFU ml1 pathogens embedded in biofilms within minutes, thus suggesting a clinical potential to decontaminate wounds, prevent surgical site infection, and promote wound healing. Sun et al.94 investigated the potential of He/O2 NTP to increase the antifungal activity of the widely used antifungicide fluconazole, amphotericin B, and caspofungin against Candida biofilms and found sessile minimal inhibitory concentrations decreased by a multiple of 2–6. These results encourage a further search for effective combinations of NTPs with traditional therapeutic means. VI. SUMMARY AND CONCLUSIONS Here, we reviewed the issues related to the role of bacterial biofilms in human diseases and recent advances in biofilm inactivation and eradication using atmospheric pressure nonthermal plasmas. The demonstrated efficiencies of NTPs for surface modification, bacterial killing, and matrix destruction pose several strategies for applying NTP for biofilm prevention and control in clinical practice. Prevention of bacterial adhesion and/or surface colonization is the most attractive strategy for reducing biofilm-associated infections. The most promising application, however, is applying NTPs in artificial materials that are modified before entering into contact with a human body. Due to their safeness, NTPs can be applied directly to human tissues that allow bacterial killing within biofilms on both abiotic and biotic surfaces such as teeth, wound surfaces, etc. The possibility not only to kill bacteria but also to destroy biofilms is an important goal as biofilm matrix includes bacterial virulence factors and toxins that can exert toxic effects even in the absence of living bacterial cells. Combinations of NTPs with other medicine that have added antibiofilm effects are very important for incorporating nonthermal plasma technologies into clinical practice. Further progress in the use of NTPs as a means of biofilm prevention and control will rely on efforts in plasma source design, fundamental analysis of plasma-biomaterial interactions, and comprehensive clinical studies. ACKNOWLEDGMENTS The authors thank V. I. Pushkareva for the unpublished image of the Lactobacillus monocytogenes biofilm. The work of the authors in the field of plasma medicine was supported by grants from the Russian Ministry of Education and Science (Grant No. 02.740.11.0310), Russian Ministry of Industry and Commerce (Grant No. 11411.1003702.13.058), and Russian Fund for Basic Research (Grant Nos. 08-0812226 and 12-08-01262). 1

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