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Potential novel antimicrobial therapies for burn wounds: peptides and cold plasma Dijksteel, Gabriëlle Sherella
2021
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Dijksteel, G. S. (2021). Potential novel antimicrobial therapies for burn wounds: peptides and cold plasma.
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Potential Novel Antimicrobial Therapies for Burn Wounds: Peptides and Cold plasma Gabriëlle S. Dijk
You are kindly invited to aaend the public defence of my PhD
thesis enntled:
Potennal Novel Annmicrobial Therapies
for Burn Wounds: PPepndes and Cold plasma
Live stream
http://www.youtube.com/
VUBeadlesOffice
Gabriëlle S. Dijksteel dijksteelgabrielle@gmail.com
Paranymphs Rabin Neslo rabinneslo@hotmail.com
Odaily Augustuszoon oodaily.augustuszoon@gmail.com Wednesday 24 March 2021 at 11:45
INVITATION
Potential Novel Antimicrobial Therapies for Burn Wounds: Peptides and Cold plasma
Gabriëlle S. Dijksteel
This PhD thesis was embedded within Amsterdam Movement Sciences research institute at the Department of Plastic, Reconstructive and Hand Surgery, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands.
Financial support for research on peptides was provided by the Ministry of Economic Affairs through two public–private partnership (PPP) allowances, made available by Health- Holland and Top Sector life Sciences & Health. One of the PPP allowances was co-funded by the Dutch Burns Foundation, Madam Therapeutics B.V., Avivia B.V., Leiden University Medical Center, Amsterdam University Medical Center and the Association of Dutch Burn Centers (LSHM17078-SGF) and the other PPP allowance was co-funded by the Dutch Burns Foundation, Madam Therapeutics B.V., Mölnlycke Health Care AB, Leiden University Medical Center, Amsterdam University Medical Center and the Association of Dutch Burn Centers (LSH-TKI40-43100-98-017). Research on cold plasma was financially supported by two grants from i) the Dutch Burns Foundation (14.104) and ii) a translational research program subsidized by ZonMw (95104007) and the Dutch Burns Foundation.
ISBN/EAN: 978-94-6423-129-8 Cover: Michalda S. Dijksteel Lay-out: Gabriëlle S. Dijksteel
VRIJE UNIVERSITEIT
Potential Novel Antimicrobial Therapies for Burn Wounds: Peptides and Cold plasma
ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam,
op gezag van de rector magnificus prof.dr. V. Subramaniam, in het openbaar te verdedigen ten overstaan van de promotiecommissie
van de Faculteit der Geneeskunde op woensdag 24 maart 2021 om 11.45 uur
in de aula van de universiteit, De Boelelaan 1105
door
Gabriëlle Sherella Dijksteel geboren te Paramaribo, Suriname
promotoren: prof.dr. E. Middelkoop dr. M.W. Ulrich copromotor: dr. B.K.H.L. Boekema
promotiecommissie: prof.dr. P.P.M. van Zuijlen dr. W. van Wamel dr. A. Sobota prof.dr. W. Bitter prof.dr. L.G. Visser
List of contents
Chapter 1: General introduction
Chapter 2: Review: Lessons learned from clinical trials using antimicrobial peptides (AMPs)
Chapter 3: SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides
Chapter 4: Potential factors contributing to the poor antimicrobial efficacy of SAAP-148 in a rat wound infection model
Chapter 5: The functional stability, bioactivity and safety profile of synthetic antimicrobial peptide
SAAP-148
Chapter 6: Safety and bactericidal efficacy of cold atmospheric plasma generated by a flexible surface Dielectric Barrier Discharge device against Pseudomonas aeruginosa in vitro and in vivo
Chapter 7: General discussion Chapter 8: Summary/samenvatting Appendixes
Abbreviations Publications Training program Curriculum Vitae Dankwoord
9 33 63 77
99
119
139
157 163 166 168 169 171 172
Chapter 1
General introduction
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Burn wounds
Epidemiology of burns
Each year approximately 750 burn patients are admitted to a specialized burn center in the Netherlands [1]. Of these patients, about 96% survives but can be faced with an impaired quality of life due to burn-related complications such as disabilities, immobilization, disfigurements, itching and post-traumatic stress disorder [1–3]. The mortality rate, of approximately 4%, is caused by the burn injury and its acute complications such as local and systemic inflammation, loss of fluids (hypovolemia) and proteins (hypoproteinemia), breathing difficulties, bacterial wound colonization and sepsis, which may lead to organ failure [4]. To minimize morbidity and mortality, severely burnt patients require immediate specialized medical care [5]. The medical care for burn patients has improved over the past decades, nevertheless, sepsis and wound colonization by bacteria continue to be a primary cause for increased morbidity and mortality rates in burn patients [6,7].
Immediately after burn injury the patient’s flora may colonize the wounds. These bacteria often originate from endogenous skin, and the gastrointestinal and respiratory tract.
Additionally, nosocomial bacteria are commonly transmitted to burn patients via the air or hands of the health care personnel during e.g. wound dressing procedures and topical treatment of burns [8–10]. Studies show that gram-positive bacteria such as Staphylococcus aureus first colonize burns within 48 h after injury, which is often replaced by gram-negative bacteria like Pseudomonas aeruginosa within one week [11,12]. In general, the most prevalent bacteria to colonize burns are S. aureus, Acinetobacter baumannii, P. aeruginosa and Enterobacter species. The prevalence of the type of bacterium is, among others, dependent on the geographic location. In the Dutch burn centers [13], the most prevalent bacterium is S. aureus but in South African [14], Singaporean [15], Chinese [16] and Turkish [17] burn units the most prevalent bacterium is A. baumannii. P. aeruginosa is most prevalent in Iran [18], Palestine [19] and Romania [20].
A study from Poland reported that wound colonization by P. aeruginosa or A. baumannii is associated with an increased risk of mortality due to sepsis in severely burnt patients [21].
Known risk factors for bacterial colonization are advanced age, a total body surface area (TBSA) of >20% affected by the burn, high numbers of surgical interventions, admission to the intensive care unit, antibiotic usage, long hospital stay and underlying diseases [1,22].
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Pathophysiology of burns
Burns initiate local and systemic inflammatory responses, which could have detrimental effects on the burn wound healing but also on several distant organs [24–26]. These responses involve excessive activation of C-reactive protein and complement, extended wound infiltration by high numbers of neutrophils and macrophages, and the prolonged release of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α, as well as reactive oxygen species (ROS) [27–29]. These responses persist because of the attenuated production of anti-inflammatory mediators such as transforming growth factor-b (TGF-b) that promotes wound healing [30–32]. As a result of the local inflammatory response to burns, a systemic inflammatory response is initiated, disturbing cardiovascular, respiratory, metabolic and immunological systems, which eventually may cause organ failure [24].
Burns can be characterized by three zones i.e. the zone of coagulation, stasis and hyperaemia (Figure 1) [33]. The outermost zone of hyperaemia is characterized by increased perfusion and inflammation, leading to redness of the skin. This zone will most likely recover, unless there is sepsis or prolonged hypoperfusion [33]. The surrounding zone of stasis is characterized by decreased perfusion. Survival or necrosis of this tissue is dependent on the wound environment. The zone of coagulation at the center of the wound consists of necrotic tissue, known as eschar, which is generally excised to allow wound healing to take place. The presence of eschar can increase the risk for bacterial colonization because eschar can serve as a nutrient source for bacteria [34].
Figure 1: Three zones of burn wounds
Burns can be characterized by three zones i.e. the zone of coagulation, stasis and hyperaemia [33]. Depending on the wound environment, the zone of stasis may survive or become necrotic tissue. This illustration was created with BioRender.com.
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Bacteria in burns
Colonization of burns
A study by Dokter et al. shows that approximately 60% of burn patient samples are “sterile”
or “non-suspicious”. Approximately 40% are commonly colonized by one or more pathogenic bacteria such as S. aureus (40%), Streptococci (30%), P. aeruginosa and other non-fermentative species (19%), Escherichia coli, Proteus, Klebsiella and Enterobacter species (each approximately 5%) [13]. Isolates resistant to antibiotics were also found in samples from burn patients and included methicillin-resistant S. aureus (MRSA), methicillin- resistant coagulase-negative Staphylococci, vancomycin-resistant Enterococci, and gram- negative bacteria that possessed one or several types of b-lactamases causing multi-drug resistance (MDR) to b-lactam antibiotics [13,35]. Yeast and fungi (2.3%) are rarely encountered but may also colonize burns [13].
Bacterial wound colonization can lead to biofilm formation and local or systemic infection when the immune response fails or topical treatment is not effective [36]. Biofilms are characterized by highly specialized bacterial communities that are enveloped in a self- produced matrix, which acts as a protective shell against various antimicrobial agents. To form a biofilm, bacteria bind to the wound bed, proliferate to form a thin layer and emit chemical signals to communicate between cells [37], known as quorum sensing [38]. Next, bacteria produce an extracellular matrix and expand to form microcolonies. At the final stage, bacteria disperse from the biofilm and colonize a new surface (Figure 2) [39].
Colonization of tissue with more than 105 bacteria per gram of tissue is usually considered to be infected [40]. During infection, bacteria spread to the surrounding and deeper tissue, causing further tissue damage and triggering inflammatory responses.
Persistent inflammation
The healing process of burn wounds discriminates three phases, i.e. the inflammation, proliferation and maturation phase that overlap in an orderly manner in time [41,42].
Bacterial colonization affects this timely ordered scheme because it lengthens the inflammation phase [43]. For example, during early biofilm formation, neutrophilic polymorphonuclear leukocytes (PMNs) attack opportunistic bacteria, such as S. aureus and
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As a consequence, the destruction of host cell membranes and proteins by ROS could be prolonged as immune cells make new efforts to eradicate bacteria using ROS. Furthermore, extracellular deoxyribonucleic acid (eDNA) of particularly P. aeruginosa biofilms activates neutrophils and macrophages, and serves as a major pro-inflammatory component [49].
The production of pro-inflammatory cytokines, such as IL-1b, IL-6 and TNF-α, promotes immune dysfunction [50,51]. Additionally, these cytokines may promote bacterial growth, which leads to the development of persistent and chronic infections [52].
Bacterial colonization may also affect the proliferation and maturation phase of wound healing. Marano et al. showed that bacterial metabolites could inhibit keratinocyte migration and degrade dermal proteins, thereby decreasing the rate of re-epithelialization [53]. Additionally, Muller el al. showed that pyocyanin, a toxin derived from P. aeruginosa, induced persistent oxidative stress and premature senescence in fibroblasts, which resulted in impaired wound healing [54]. Furthermore, as a result of the prolonged inflammatory response, excessive fibroblast proliferation and collagen deposition could be promoted.
This results in the formation of disorganized and excessive scars, such as hypertrophic scars.
Figure 2: Biofilm life cycle
Single bacterial cells adhere to the surface and proliferate to form a monolayer. Thereafter, bacteria emit chemical signals to communicate between cells and produce an extracellular matrix (ECM). They expand to form microcolonies, i.e. a mature biofilm and finally, they disperse from the biofilm and colonize a new surface [39].
This illustration was created with BioRender.com.
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Antimicrobial treatment for burns
Antibiotics
To prevent and manage bacterial colonization in burn patients several strategies and evidence-based guidelines are generally accepted, i.e. hand hygiene protocols, environmental disinfection protocols, monitoring of bacterial presence by routinely taking swabs or biopsies for culture and the creation of an antibiotic resistance profile (antibiogram) to assist the treatment plan [55]. However, the use of antibiotics remains controversial due to the emergence of MDR bacteria [56]. Currently, MDR is a global health concern because bacteria can spread worldwide and cause lethal infections for which there is no effective treatment yet (Figure 3) [56]. The widespread and inappropriate use of antibiotics at sub-inhibitory concentrations has exacerbated the MDR problem [57].
Therefore, a proper regimen for effective topical and systemic antibiotic therapy in burns is essential.
The mortality rate of severely burned patients is significantly reduced by selective intestinal decontamination (SID) without inducing bacterial resistance [58,59]. SID is used as prophylactic therapy and consists of parenteral and enteral antibiotics with limited anaerobicidal activity to prevent or minimize the impact of endogenous bacteria. A SID regimen commonly includes the intravenous administration of cefotaxime for 4 days and the digestive administration of polymyxin E, tobramycin and amphotericin B [59,60].
Thereafter, an empirical antibiotic regimen consisting of vancomycin, a b-lactam antibiotic and a fluoroquinolone or aminoglycoside is often considered. Anti-pseudomonal carbapenem is the agent of choice in facilities with high incidence of b-lactamase producing bacteria [61].
Antibiotics are systemically administered to burn patients suffering from pneumonia, wound infection and sepsis [62]. However, systemically administered antibiotics may not reach the infected wound at an effective dose due to the compromised blood circulation in burns [63,64]. Therefore, burn patients may also receive topical antibiotics such as bacitracin, polymyxin B and neomycin alone or in combination, or mupirocin. Mupirocin is a topical antibiotic that can suppress colonization by MRSA [65]. It is mainly used to treat skin and nasal colonization by S. aureus [66,67]. However, its selective mode of action led to the emergence of mupirocin-resistant bacteria with mutations commonly located in the
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Antiseptics
Antiseptics such as hypochlorous solution [72], povidone-iodine [73,74], chlorhexidine [75,76] and peroxide agents [77,78] have a broad spectrum of activity and act through multiple mechanisms simultaneously. As a consequence, bacteria do not develop resistance to antiseptics as readily as to antibiotics. However, many antiseptics are cytotoxic for human keratinocytes and fibroblasts, and may impair wound healing [79,80]. In general, the required concentration to effectively eradicate bacteria and to avoid cytotoxicity is unknown for many antiseptics. Therefore, the use of antiseptics is limited to irrigation or wound cleaning.
Figure 3: Prevalence of antimicrobial resistant S. aureus in 2018
According to the data from the European Centre for Disease Prevention and Control (ECDC), the number of resistant S. aureus isolates in 2018 increased in several European countries as compared to that of 2017 [81]. In the Netherlands, the number of resistant isolates remained relatively low. However, traveling Dutch citizens may return with resistant microorganisms, suggesting an increase in the number of resistant pathogens in the Netherlands as well.
Topical antimicrobials
Topical antimicrobial agents commonly used in burn wound care are fusidic acid, Prontosan®, and silver-based agents such as silver nitrate, silver sulfadiazine (SSD) and
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systemically to treat S. aureus colonization [71,82]. Monotherapy with fusidic acid is not recommended due to the likelihood of resistance development [83,84]. Resistance rates of approximately 5% have been reported for fusidic acid monotherapy versus 1% for combination therapy with conventional antibiotics [85]. There is a strong suggestion that combination therapy of fusidic acid and antibiotics reduces resistance selection [86,87].
Additionally, combination therapy may result in a more effective bacterial killing due to synergy [88]. Fusidic acid in combination with rifampin is most common [89,90] but no particular antibiotic appears to be the best at preventing the selection for resistance [85].
Prontosan® is used for the prevention and removal of biofilms [91,92]. It contains betaine, which is a detergent required to penetrate biofilms and to remove wound debris [93,94]. It also contains polyhexanide, which is effective against a wide range of gram-positive and gram-negative bacteria [92,95–97]. No evidence of bacterial resistance development has been demonstrated for Prontosan® but in some cases, patients report allergic reaction such as itching and rashes [98,99].
As for the silver-based agents, SSD has long been considered as the gold standard for the treatment of burns [100]. Bacterial resistance to silver is uncommon, presumably because silver acts through multiple mechanisms to eradicate bacteria. These include the inhibition of enzymes necessary for metabolism of the microorganism, disruption of the cell membrane and interference with DNA and RNA preventing replication of bacteria [101–
103]. However, poor penetration into burn eschar and/or biofilm, and cytotoxicity for skin cells resulting in a delayed wound healing, are the main drawbacks of silver-based agents [104–106]. To improve wound healing outcomes, SSD could be used in combination with cerium nitrate. Cerium nitrate has bacteriostatic effects and may stabilize the burn eschar through the reduction of bio-burden and inflammatory cytokines [107]. The addition of cerium nitrate to SSD results in a relatively tough, leather-like eschar, which inhibits infection and long-term adherence of bacteria to the wound [108]. As a result, surgeons may perform late excisions of eschars followed by auto-grafting in burn patients where early excisions of large areas did not prove to be beneficial for survival [108]. Ultimately, this treatment strategy may improve the wound healing and decrease the risk of bacterial colonization and infections. To validate these beneficial outcomes, several in vivo studies have been performed but concluded that there is no clear-cut superiority for the addition of cerium nitrate to SSD over SSD only [109–111]. Additionally, Rashaan et al. concluded that non-silver containing treatments may be preferred over SSD [112]. Hence, there still is
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Potential new antimicrobial therapiesAntimicrobial peptides Definition and structure
Antimicrobial peptides (AMPs) are a diverse class of naturally occurring molecules that are produced by epithelial cells, phagocytes, lymphocytes and other cell types [113]. They act as the first line of innate immune defense against various pathogens such as bacteria, fungi and viruses [114]. Efficacy of AMPs is dependent on the length, sequence and conformation. Despite these structural differences, AMPs have some common features such as an amphipathic structure and a net positive charge ranging from +2 to +11.
Therefore, they are generally referred to as cationic host defense peptides or cationic amphipathic peptides but anionic AMPs also exist. There is consensus that the positive charge of the AMP is essential for initial binding to the pathogenic membrane/proteins and that hydrophobicity is needed for insertion into the membrane of the microorganism.
Mode of action
Based on the mode of action, AMPs are classified into two families, i.e. i) membrane disruptive (barrel stave, toroidal, sinking raft and micellar models) and ii) non-membrane disruptive (inhibition of protein or DNA synthesis and neutralization of toxins) AMPs [115,116]. Besides direct antimicrobial activity, AMPs can display several immunomodulatory properties such as the differentiation and recruitment of immune- competent cells, the modulation of cytokine release and the acceleration of wound healing [117,118]. Additionally, AMPs can display synergic interactions with conventional antibiotics [119–121].
Bacterial resistance and cytotoxicity of AMPs
Due to the AMPs’ multi-hit, nonspecific and rapid mode of action, the development of bacterial resistance to AMPs has generally been considered to be unlikely [118,122,123].
However, several AMPs, including the human cathelicidin LL-37, human β-defensins 2 and 3 and lactoferricin B, have been associated with bacterial resistance due to the phenotypic characteristics of bacteria such as the slow growth rate, diminished transmembrane potential and altered metabolism [124–128]. Hence, the risk of resistance should be carefully investigated.
To treat bacterial infection in humans, the effective AMP concentration should at least be
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exhibit low affinity for the membrane of mammalian cells. However, several studies demonstrate that AMPs may induce hemolysis and apoptosis in mammalian cells [129,130].
Other studies show that the level of cytotoxicity depends on the structure of AMPs i.e. the positive charge, α-helical contents, the hydrophobicity and amphipathicity of the AMP [131,132]. Moreover, there is a correlation between the ratio of aromatic residues such as tryptophan to cationic residues and a high degree of hemolysis and cytotoxicity [133,134].
Nonetheless, cytotoxicity of AMPs is concentration dependent. Low AMP concentrations show no or limited cytotoxicity, while still being effective against bacteria [135].
Limitation of AMPs
The development of AMPs suitable for clinical use remains a challenging task. For example, antimicrobial activity of AMPs is highly dependent on environmental conditions.
Physiological salt and serum conditions may reduce the effective concentration of available AMPs to eradicate bacteria. Therefore, the ability to achieve high bactericidal activity with AMPs in a wound environment is often questioned [136]. To overcome this limitation, detailed pharmacokinetic/pharmacodynamic studies are required to develop AMPs with low affinity for e.g. serum proteins, salts and host cells.
Applications of AMPs
AMPs are considered potential therapeutic agents for the treatment of e.g. surgical site/soft tissue infections [135,137], urinary tract infections [138], Clostridium difficile disease [139], pneumonia [140] and biofilm inhibition [141,142]. Currently, D2A21 is the only AMP under clinical evaluation that specifically targets burn wound infections. D2A21 significantly reduced the bacterial count in burn wound infections in rats and displayed a favorable safety profile in vitro and in vivo [143,144]. Similarly, the AMP PXL150 effectively eliminated P. aeruginosa and displayed a favorable safety profile following repeated administration systemically and locally in rats and rabbits, respectively [135,145]. Another pre-clinical study of AMP SAAP-148 previously received attention due to its effectivity against MDR ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P.
aeruginosa, and Enterobacter species) in vitro and in in vivo mouse models [146].
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Cold gas plasma
Definition and classification
Gas plasma has been used for sterilization purposes of medical devices and dietary products [147]. It could be used to inactivate pathogenic bacteria in (burn) wounds as well. Plasma is ionized gas, which is commonly referred to as the fourth state of matter. It can be encountered in the sun, stars, solar winds, lightning in the sky’s but also in rocket exhausts, TV screens and fluorescent lamps. The ions in plasma coexist with free electrons, reactive species, radicals, electrical fields and ultraviolet (UV) radiation. Based on the relative temperature of these chemical species, plasmas are classified as thermal and non-thermal or cold plasma (CP). The temperature of the electrons, ions and other molecules are in equilibrium in thermal plasma but the temperature of the electrons is much higher than that of the ions and other molecules in CP. These plasmas can be artificially generated in the laboratory. To generate thermal plasma, a gas is subjected to a relatively high temperature (10-100 eV) and pressure (>1 atm), whereas for CP a gas is subjected to a strong electromagnetic field at atmospheric or vacuum pressure. The application of a strong electromagnetic field to a gas at atmospheric pressure is commonly used in plasma devices like plasma jet and dielectric barrier discharge (DBD) to generate CP. The type of gas used for the generation of CP generally influences the composition and yield of CP-induced reactive species [148,149]. Noble gasses such as helium (He), neon (Ne) and argon (Ar) have been used to generate CP [150–152]. However, the energy to ionize these gases is higher than for oxygen (O2), nitrogen (N2) and atmospheric air. Atmospheric air consisting of O2, N2 and water vapor is considered an excellent gas for the generation of CP [153].
Mode of action
Four possible features of CP from atmospheric air could be involved, independently or in synergy, in the eradication of bacteria, i.e. heat, UV light, electric fields, and charged and reactive species (Figure 4) [154]. Heat is produced during the generation of CP due to the collision of electrons, and the subsequent excitation, ionization and dissociation processes of the gas particles [155]. Depending on several factors, such as the frequency of the discharge and the treatment time, the temperature of CP may raise to approximately 120°C [156]. Nevertheless, the temperature of the CP-exposed sample may not significantly raise above 40°C [156,157]. This temperature is not sufficient to eradicate bacteria.
Temperatures of 60°C for a duration of 30 min were required to denature (membrane) proteins and kill P. aeruginosa [158,159]. UV light of wavelengths between 240 and 280 nm is germicidal and may cause distortion of DNA strands [160]. CP from atmospheric air commonly emits UV light of wavelengths above 280 nm and makes a relatively minor
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contribution to bacterial killing [161,162]. High electrical fields cause a change in trans- membrane potential, which initiates transformation of membrane structure and ultimately induces cellular death [163]. Bacterial killing by an electrical field is unlikely for CP, because the electrical field exponentially reduces to a fraction of its value over a very short distance of less than 300 µm [164,165]. To contribute to killing, bacteria should be positioned within this distance from the electrical field.
It is generally accepted that charged and reactive species such as electrons, atomic oxygen (O1), ozone (O3), hydrogen peroxide (H2O2), hydroxyl (·OH), hydroperoxyl (·OOH), nitric oxide (NO), nitrite (NO2-), nitrate (NO3-) and peroxynitrite (ONOO-) radicals are responsible for bacterial killing by CP from atmospheric air [166]. However, there is still uncertainty whether reactive oxygen species (ROS) or reactive nitrogen species (RNS) are the main components in CP responsible for bactericidal activity. Kylián et al. reported that a greater amount of O2 gas in a O2/N2 gas mixture used for the generation of CP results in more shrinkage of bacterial endospores [167]. Therefore, ROS are most likely the main components in CP responsible for bactericidal activity via lipid peroxidation and a mechanism called etching [168–170]. Etching is the formation of lesions and openings in the bacterial membrane, ultimately causing leakage of bacterial cell content. The presence of RNS enhances the bactericidal activity of O2 plasma species [171–174]. High amounts of NO2- and H2O2 can form ONOO- and O2NOOH, and ultimately O2- radicals by multiple reactions [171,173]. The high abundance of such free radicals can lead to the penetration of reactive oxygen and nitrogen species (RONS) inside the bacterial wall [175], causing intracellular damage such as DNA damage and protein damage [176,177]. Additionally, the presence of RNS induces the formation of nitrous (HNO2) and nitric acid (HNO3), thereby decreasing pH [166,178]. Since the intracellular pH of bacteria plays a major role in their functioning [179], acidification may contribute to the fast bactericidal efficacy of CP [180,181]. Hence, CP from atmospheric air eradicates bacteria mainly by causing lipid peroxidation/etching, DNA and protein damage, and acidification.
Bacterial resistance and cytotoxicity of CP
Due to the nonspecific mode of action of CP, the development of bacterial resistance is not expected. The possibility for bacterial resistance to CP should not be excluded because CP
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Figure 4: Components of cold plasma [189]
Plasmas consist of reactive species, electrons, ions, heat and UV radiation, and electromagnetic fields. Of these components, the reactive species, electrons and ions are most likely responsible for bacterial killing [166]. Plasmas from atmospheric air often emit purple-blue light, which is predominantly caused by excited nitrogen molecules that return back to the ground state, i.e. the lowest energy state. The excess energy is released in the form of photons, which falls in the visible light region of the electromagnetic spectrum. This illustration was created with BioRender.com.
Limitation of CP
It remains a challenging task to demonstrate the potential benefits of CP in vivo. Efficacy is dependent on many operating and environmental factors including the treatment time, ionization energy, distance to the tissue and the presence of moisture, salts and exudate.
Previously, Kvam et al. demonstrated that higher numbers of bacteria were eradicated as the treatment time was increased [190]. Similarly, high ionization voltages were more effective against bacteria than low ionization voltages [154]. As these conditions might also cause cytotoxic effects, the balance between optimal bactericidal efficacy and cytotoxicity should be determined. Furthermore, the distance between the plasma source and tissue
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distance, reduces the concentration of H2O2 and O3 significantly [162,191]. Similarly, a high or low moisture content negatively effects the efficacy of CP [192]. Nonetheless, moisture is an important parameter for the generation of ROS such as ·OH and H2O2. Finally, the presence of salts may result in less bacterial killing [193]. Previously, Attri et al. show that the presence of NaCl in solution decreases the generation of OH radicals [194]. The presence of salts may also increase pH and/or introduce buffering effects [195,196]. This results in less bacterial killing via acidification due to a lower yield of RNS such as HNO2 and HNO3. Despite these limitations, CP displays promising bactericidal properties for clinical application in burns when operated at optimal conditions.
Application of CP
Clinical studies using plasma technology mainly focus on the eradication of bacteria in chronic wounds [197,198] because chronic wounds are easily accessible for superficial treatment using CP. Previously, Isbary et al. reported the results of a randomized controlled trial of a CP from Ar gas. This CP was safe and successfully reduced the bacterial load in chronic wounds. Pre-clinical research using CP from atmospheric air showed that CP can eradicate MRSA strains in planktonic and biofilm forms [199]. Another pre-clinical study showed that CP from atmospheric air may promote wound healing because it eradicates pathogenic bacteria that impair the healing [200]. Furthermore, Boekema et al. show that treatment with CP from a flexible surface DBD device is not only effective against pathogenic bacteria but also safe for human skin cells [195]. Such CP has potential for treating infected burns.
Thesis aims and outline
Colonization of burns by pathogenic and MDR bacteria remains a threat to the well-being of severely burned patients. There is still an urgent need for safe and effective antimicrobial treatments, which are less likely to induce bacterial resistance. Therefore, the purpose of this thesis is to investigate novel antimicrobial therapies as potential treatments for colonized burns. Both AMPs and CP show antimicrobial activity and a promising safety profile at bactericidal concentrations and settings, and duration of the treatment. Hence,
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DBD device against pathogenic bacteria; thirdly, to assess the safety of these antimicrobial strategies for human skin cells.
Currently, only a few AMPs are in clinical use. Therefore, we reviewed the literature to determine the challenges towards clinical application of AMPs (Chapter 2). Additionally, we describe various strategies that are currently available to improve AMPs for clinical use.
To determine efficacy of antimicrobial agents accurately, neutralization of residual antimicrobial activity before microbiological assessment of the number of surviving bacteria is required. We have studied the ability of sodium polyanethol sulfonate (SPS) to neutralize residual activity of highly positively charged agents (Chapter 3). Subsequently, we used SPS to determine the in vivo efficacy of SAAP-148 in a partial-thickness wound infection model in rats (Chapter 4). In this model, the efficacy of SAAP-148 against MRSA was significantly reduced as compared to in vitro conditions. Therefore, we investigated several factors that may have affected the in vivo bactericidal activity of SAAP-148. We also determined the functional stability and activity of SAAP-148 in biologically relevant environments in vitro (Chapter 5). Relatively high antimicrobial concentrations were required to effectivity eradicate MRSA in biological environments. Subsequently we determined the in vitro and ex vivo cytotoxicity of SAAP-148 for human skin cells using high antimicrobial concentrations.
The potential of CP as an alternative therapy for antibiotics or as supplementary agent to eradicate MDR bacteria is still under investigation. We assessed the in vivo efficacy of CP using a partial-thickness wound infection model in rats (Chapter 6). We also studied additional safety aspects such as the potential of CP to induce mutations and apoptosis in eukaryotic cells.
The results obtained in these studies are discussed in Chapter 7 and an English and Dutch summary is provided in Chapter 8.
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