Special Issue: Plasma Biotechnology

Review

Cold Plasmas for Biofilm Control:

Opportunities and Challenges
Brendan F. Gilmore,1,*,@ Padrig B. Flynn,1 Séamus O’Brien,1 Noreen Hickok,2 Theresa Freeman,2 and
Paula Bourke3

Bacterial biofilm infections account for a major proportion of chronic and Highlights
medical device associated infections in humans, yet our ability to control them Biofilms are implicated in around 65%
of all chronic human infections, includ-
is compromised by their inherent tolerance to antimicrobial agents. Cold atmo-
ing those associated with indwelling
spheric plasma (CAP) represents a promising therapeutic option. CAP treat- medical devices such as catheters
ment of microbial biofilms represents the convergence of two complex and prostheses. Biofilm infections are
often asymptomatic between exacer-
phenomena: the production of a chemically diverse mixture of reactive species bations and challenging to detect and
and intermediates, and their interaction with a heterogeneous 3D interface effectively treat using conventional
created by the biofilm extracellular polymeric matrix. Therefore, understanding antibiotics and antimicrobial agents.

these interactions and physiological responses to CAP exposure are central to CAP provides an effective multimodal,
effective management of infectious biofilms. We review the unique opportu- multitarget approach for controlling
microbial biofilms.
nities and challenges for translating CAP to the management of biofilms.
Biofilms express a complex extracellu-
Challenges in Biofilm Control lar matrix of polymeric substances that
Biofilms (see Glossary) are complex consortia of microbial cells (bacteria, archaea, and fungi) may attenuate the antimicrobial effi-
that are attached to a substratum (biotic/abiotic surfaces and/or each other) and embedded cacy of CAP via interactions with
CAP-generated RONS.
within a matrix of self-produced or acquired extracellular polymeric substance (EPS),
including polysaccharides, DNA, protein, and lipids [1–5]. Biofilms are characterized by signifi- Biofilm tolerance to CAP is variable
cantly elevated tolerance to antimicrobial agents, attributed to restricted penetration of anti- between species and between strains
microbials, altered (decreased) growth rates and transcription within the biofilm, formation of of the same species, which may be
due to production of EPS, RONS-
persister cells, and quorum sensing-controlled tolerance/protective mechanisms [6,7] (Fig- detoxifying enzymes, or acquired tol-
ure 1). Consequently, the effectiveness of conventional antibiotics, biocides, and normal erance to physiological RONS during
immune clearance is severely limited and biofilms are often implicated in chronic, persistent, chronic infections.
and recurrent infections [1–4]. Furthermore, the structure of polymicrobial biofilms facilitates
transfer of antimicrobial resistance, compounding the problem. Thus biofilm formation is
now widely recognized as a major virulence determinant in a wide range of chronic infections 1
Biofilm and Pharmaceutical
[2]. The importance and ubiquitous distribution of biofilms (Box 1) as a distinct and predominant Microbiology Research Group, School
microbial phenotype in chronic and recurrent infections (including those on medical devices) of Pharmacy, Queen’s University
Belfast, 97 Lisburn Road, Belfast, BT9
and in environmental niches and engineered systems has prompted a rethink in how we 7BL, UK
discover and develop new antibiofilm therapeutic approaches. 2
Department of Orthopaedic
Research, Sidney Kimmel Medical
College of Thomas Jefferson
While antibiotic resistance remains a major global issue, in the context of biofilm control, an University, Jefferson Medical College,
important distinction between antimicrobial (antibiotic or biocide) resistance and antimicrobial 1015 Walnut Street, Suite 501,
tolerance must be recognized. Antimicrobial resistance arises from irreversible, heritable Philadelphia, PA 19107, USA
3
Plasma Research Group, School of
changes to the microbial genome (and is therefore genotypic); antimicrobial tolerance is a Food Science and Environmental
phenotypic and reversible, and enables the microorganism to withstand and survive antimi- Health, Dublin Institute of Technology,
crobial treatments [7,8]. One of the greatest threats to our ability to control infectious diseases is Marlborough Street, Dublin 1, Ireland
@
Twitter: @BrendanFGilmore
the lack of an effective developmental platform for antibiotic discovery [9]. In the absence of a
sustainable pipeline of antibiotics, the emergence of antimicrobial resistance continues *Correspondence:
unchecked, undermining the efficacy of the remaining armamentarium of clinically useful drugs. b.gilmore@qub.ac.uk (B.F. Gilmore).

Trends in Biotechnology, June 2018, Vol. 36, No. 6 https://doi.org/10.1016/j.tibtech.2018.03.007 627

© 2018 Published by Elsevier Ltd.
The growing prospect of a postantibiotic era, general pessimism surrounding new antibiotic Glossary
discovery, and a growing urgency as annually, increasing numbers of patients succumb to Aerobic respiration: generation of
infections caused by resistant microbial pathogens, has prompted renewed interest in alter- respiratory energy whereby oxidation
natives to conventional antibiotics [7,10]. (of, for example, glucose) occurs in
the presence of oxygen and where
O2 acts as the terminal electron
One such nonantibiotic option is the use of nonthermal or CAP for the eradication and control of acceptor.
both acute and chronic biofilm infections. Plasma medicine is as nascent research field that Anaerobic respiration: respiratory
energy generation in which electron
may be uniquely positioned to address the particular challenges associated with controlling
acceptors other than oxygen are
infectious biofilms. Is it possible to translate the increased understanding of biofilms, gained utilized. Occurs in the absence of
over the past four decades (in particular the mechanisms governing tolerance to antimicrobial oxygen.
challenge), to optimize cold plasmas to control these complex microbial communities? Antimicrobial resistance:
genetically acquired ability of bacteria
to resist the effects of antimicrobial/
Plasma Medicine: A New Frontier in Biofilm Control antibiotic drugs. Resistant
The efficacy of CAP in controlling bacterial biofilms has been widely reported in the plasma microorganisms are not killed or
medicine literature, (for recent reviews, see [11,12]) and is the subject of intensive research. Topical inhibited in the presence of certain
antibiotic/antimicrobial agents.
applications have been developed using a range of plasma sources for chronic wounds, mucous
Antibiotic resistance is encoded by
membranes, and the oral cavity. While a thorough discussion of the plasma sources used within several genes within the bacterial
the wider plasma medicine field is beyond the scope of this review, a recent perspective describes genome, which may be transferred
them in more detail [13]. The 2012 Plasma Roadmap outlined the key challenges within the field of between bacteria.
Antimicrobial tolerance: reversible
low-temperature plasma physics and technology, and its various subfields including plasma phenotypic state that confers a
medicine [14]. The updated 2017 Plasma Roadmap redefines the key challenges for plasma general lack of susceptibility to
medicine; particularly the potential barriers to translation of cold plasma to clinical applications antimicrobial challenge, enabling the
which includes: (i) the effects of gas phase interactions with hydrated biological matrices, leading to microorganism to withstand and
survive antimicrobial treatments.
plasma activated media; (ii) defining plasma dose; (iii) tissue-specific effects on the flux of species Bioelectric effect: electrical
delivered; and (iv) modulation of the immune response [15]. enhancement of efficacy of
antimicrobial agents against biofilm
bacteria.
A wide range of nonthermal atmospheric pressure plasma devices, based mainly on plasma
Biofilm: aggregate of
jets or dielectric barrier discharge (DBD) configurations, have emerged over the past two microorganisms in which cells that
decades, which offer the possibility of either direct or indirect plasma biofilm exposures are frequently embedded within a
(Figure 2). Direct exposure has been defined as either exposure of the sample directly under self-produced matrix of EPSs adhere
to each other and/or to a surface
the plasma plume discharge [16,17] or where the substrate (e.g., tissue) acts as an electrode
(IUPAC definition). Biofilms exhibit an
that participates in creation of the plasma discharge [18]. In contrast, indirect exposure altered growth rate and transcription,
describes sample exposure whereby the sample placement is outside the plasma discharging and elevated antimicrobial tolerance
area [16–18]. Therefore, in the case of direct plasma exposure, the biofilm is exposed to high compared to free-floating (planktonic)
bacteria of the same species.
electric fields (especially in the case of DBD), charged particles, and UV [16–20]. While
Extracellular DNA (eDNA): DNA
atmospheric pressure plasmas are unlikely to produce UV doses capable of contributing that originates from the bacteria
significantly to bacterial inactivation [19,20], high electric fields may contribute to bacterial within the biofilm and forms an
inactivation [18,19]. The bioelectric effect, the electrical enhancement of antimicrobials important structural component of
the biofilm matrix EPS. It may be
against biofilm bacteria is well established, especially in the medical devices field [21]. Intense important for biofilm stabilization and
nanosecond-pulsed electric fields have been shown to alter the physiology of mammalian and antimicrobial tolerance by increasing
bacterial cells [22]. In addition, Stoodley and colleagues have demonstrated that contraction the anionic character of the biofilm
and expansion of biofilms occurs under the influence of electric fields (voltage applied with matrix, thus binding cationic
antimicrobial agents.
oscillating polarity), leading to increased susceptibility of the biofilm to antibiotics [23]. The role Extracellular polymeric substance
of charged particles in direct exposure inactivation of bacteria has also been described, (EPS): the major structural and
whereby charge accumulation on the bacterial membrane and the resulting electrostatic force functional components of microbial
biofilms; they comprise a variety of
overcomes the tensile strength of the membrane, leading to rupture [24,25]. However, Lu and
high-molecular-weight polymers (for
colleagues have demonstrated that excited N2, N2+, and He* are not expected to play a example, polysaccharides and
significant role in bacterial inactivation [19]. The combined effect of UV, electric fields, and eDNA). EPS forms a 3D, viscoelastic
charged particles may prove synergistic in inactivation of bacteria during direct plasma hydrated and often charged matrix in
which microorganisms are
exposures [26].

628 Trends in Biotechnology, June 2018, Vol. 36, No. 6

In indirect exposure scenarios (Figure 1) CAP generates a dynamic and diverse array of reactive embedded. The EPS is central to
oxygen species (ROS), which include atomic oxygen (O), superoxide (O2 ), hydroxyl radicals determining the physicochemical and
metabolic profile of the biofilm.
( OH), singlet oxygen (1O2 ), ozone (O3), and hydrogen peroxide (H2O2); and reactive nitrogen Microaerobic respiration:
species (RNS), which include nitric oxide (NO ), nitrogen dioxide (NO2 ), nitrite (NO2 ), nitrate generation of respiratory energy only
(NO3 ), and peroxynitrite (ONOO ) [27–29]. While CAP-induced biological effects are thought in the presence of oxygen at lower
to be due to the predominant reactive oxygen and nitrogen species (including organic radicals) oxygen tensions than in air, due to
the microorganisms limited capacity
which are generated in, and transferred to, the hydrated matrix of the biofilm [30], the overall to respire or presence of oxygen-
composition of reactive species produced can vary significantly depending on how CAP is labile cellular biomolecules.
generated (the source configuration, feed gas, and gaseous environment) and nature of the Pathogen: a specific causative
agent, especially a microorganism, of
liquid or biological interface with which the plasma interacts. Biofilms represent a complex,
infection or disease.
hydrated biological interface containing water, inorganic ions, and organic molecules [poly- Persister cells: phenotypic variants
saccharides, extracellular DNA (eDNA), surfactants, lipids, and proteins] [4], dispersed within a microbial population, which
throughout with bacterial microcolonies. The precise composition of plasma-derived reactive are dormant and transiently tolerant
to stress. Persister cells neither grow
species that diffuse into the biofilm are therefore dependent on the interaction of reactive
nor die in the presence of lethal
oxygen and nitrogen species (RONS) with matrix components, interaction with dissolved concentrations of antimicrobial
solutes in the hydrated matrix, and affected by alterations in pH and oxygen concentration agents or in response to lethal stress
within the biofilm. that renders the majority of the
population nonviable.
Quorum sensing (QS): mechanism
A number of studies have examined the depth of penetration of plasma-derived active species of bacterial cell-to-cell
into biofilm matrices. Xiong and colleagues have demonstrated that a He/O2 plasma jet communication that involves the
completely inactivated Porphyromonas gingivalis biofilms to a depth of 15 mm [31], while production, release, detection, and
transcriptional response to small
plasma penetrated Enterococcus faecalis biofilms 25.5 mm in depth [32]. The efficacy of an diffusible molecules called
argon plasma was shown to be reduced in proportion to Pseudomonas aeruginosa PA103 autoinducers. This cell-to-cell
biofilm thickness [33], while Alkawareek and colleagues reported (He/O2) plasma penetration communication mechanism permits
into P. aeruginosa PAO1 biofilms between 40 and 80 mm in depth [34]. However, these studies population density-dependent gene
regulation and transcription, and
were limited by the final thickness of the cultured biofilms, rather than demonstrating definitive population-wide response to
plasma penetration limits within the biofilm, which are likely to be influenced significantly by not changes in the bacterial population.
only the extent of the matrix (biomass) but also its molecular composition (influencing the nature Viable but nonculturable (VBNC):
bacterial cells that exhibit metabolic
of the interaction between matrix and reactive species), which may vary between species and
activity but cannot be cultured on
strains of bacteria. In a recent study, around 5% of ROS and 80% RNS produced by a He/O2 laboratory media on which they
plasma jet penetrated through 500 mm of biological tissue, with some RONS capable of would normally grow and divide.
penetrating up to 1.25 mm. This indicates that absolute penetration depths of RONS into Therefore, VBNC cells are not
detected by routine laboratory
biofilm matrices are likely to be underestimated by current studies [35].
culture methods but may retain
pathogenicity. The VBNC state may
Additionally, significant challenges exist to translating data from in vitro, ex vivo, or in vivo animal be induced by adverse conditions,
studies of CAP biofilm control to clinical applications. There exists an overall general lack of such as environmental stress or
antimicrobial insult. VBNC is distinct
standardized tests to evaluate new antibiofilm technologies, antibiotics, biocides, and anti-
from dormancy, which is a reversible
biofilm agents despite the development of a small number of standardized methods [including state of metabolic shutdown.
ASTM International (formerly ‘American Society for Testing and Materials’) methods for biofilm
biocide susceptibility testing] [36], and a lack of agreement as to the clinical benefit of biofilm
susceptibility testing [37]. Recently, the biofilm research community has developed guidelines,
through the ‘minimum information about a biofilm experiment’ (MIABiE) initiative [38]. These
guidelines aim to simplify the exchange, interpretation, and comparison of biofilm experimental
data, by encouraging researchers in the field (partly by enforcing the use of controlled
vocabularies and adherence to minimum information standards) to document their experi-
ments comprehensively and unambiguously. The benefits of such guidelines within the biofilm
field, where high-throughput screening and omics data sets are commonplace, are clear. Given
the complexity, scale, and variability of experimental protocols and data generated within the
highly interdisciplinary plasma medicine field, the research community should consider
generating similar guidelines (for example, minimum information about a plasma medicine

Trends in Biotechnology, June 2018, Vol. 36, No. 6 629

 Plasma
Light photons Electrons Metastables Ions Electric fields
E
hv

Neutral species
N O H

OH NOx
O2

H2O
Effluent O3 H2O2
region
N2
[NO3-]
[ONOO-]

[O2-] [H2O+]
e-(aq) [OOH-]
Biofilm interface
[NO2-] i O2

Hydrated,
nega vely
charged matrix

Cell-to-cell
signals Change in
physiology Biofilm
dispersal
eDNA
Protein
Fast
Few nutrients growers

Low O2
Alginate
Persister cells Slow growers

(See figure legend on the bottom of the next page.)

630 Trends in Biotechnology, June 2018, Vol. 36, No. 6

 Box 1. Biofilms
A biofilm can be defined as a microbially derived sessile community of cells irreversibly attached to a substratum (biotic
or abiotic), embedded within a matrix of EPS that they have produced and exhibiting an altered phenotype with respect
to growth rate and gene transcription. Biofilms represent the predominant mode of growth of bacteria in both natural
and engineered environments and in the colonization of plants, animals, and humans. In fact, the majority of chronic
human bacterial infections (estimated at 60–80%) are caused by bacterial biofilms. Biofilms provide and maintain a
unique, privileged environment that allows nutrient sequestration, facilitates cooperative activities (often controlled by
cell-to-cell communication, or quorum sensing), and permits the bacteria within to withstand and tolerate antimicrobial
challenges (by retarding penetration of antimicrobial agents), host immune clearance, and mechanical removal from a
surface. The unique environment created within the biofilm gives rise to metabolically heterogenous populations, with a
spectrum of metabolic activity and dormancy. This contributes not only to elevated tolerance to antimicrobial agents and
antibiotics, but also drives the emergence of persister cells, phenotypic variants capable of withstanding lethal stresses.
The biofilm matrix plays a central role in providing architectural and structural integrity to the biofilm, but also maintains
pH and electrochemical gradients, which ensure the heterogeneity of the microbial population within. In human health,
biofilms are frequently implicated in a diverse array of chronic, persistent infections and infections of indwelling medical
devices; from urinary catheters to prostheses. Bacteria such as Enterococcus faecium, S., epidermidis, Klebsiella
pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp. (collectively known as the ESKAPE
pathogens) alongside Enterococcus faecalis, E. coli, methicillin-resistant S. aureus (MRSA) and Streptococcus spp.
represent the most common and life-threatening causative pathogens of biofilm-associated infections.

experiment – MIAPME or minimum information about a CAP experiment – MICAPE) for

reporting on experiments on plasma–biological interactions and effects.

Biofilm Microenvironment in Chronic Infection: Preconditioning Pathogens to

RONS?
Microorganisms account for less than 10% of the dry weight of the biofilm, with extracellular
polymeric components largely accounting for the remainder [4,39]. These extracellular poly-
meric substances buffer RONS while facilitating microbial adhesion and aggregation, water
retention, confer biofilm spatial arrangement/architecture, and importantly, provide a protective
barrier against host defenses and antimicrobial agents [4]. The biofilm also contains multiple
microenvironments (hypoxic and anoxic) in which a combination of bacterial aerobic, anaer-
obic, and microaerobic respiration and metabolic pathways operate [40]. Thus to develop a
successful strategy for CAP-mediated control of biofilms in chronic infections, the bacteria
type, metabolic state and oxygen microenvironment (as well as potential reaction products
generated) must all be taken into consideration when controlling efficacy (Figure 1). Our
understanding of the biofilm–plasma interaction requires fundamental experimental studies
both in CAP physics, microbiology and meta-transcriptomics to understand the plasma-active
species driving biological phenomena within the biofilm.

An important question regarding the potential efficacy of CAP is the propensity of certain
pathogens to survive adverse conditions. For example, P. aeruginosa can withstand oxygen
limitation within the biofilm by adjusting respiration from aerobic (occurring only in the presence
of oxygen) to anaerobic (occurring in the absence of oxygen) and/or microaerobic (occurring
only in oxygen tensions lower than that of air). In particular, NO3 respiration occurs under both

Figure 1. The Plasma–Biofilm Interface. The plasma-derived reactive species that diffuse into the biofilm encounter a hydrated, cationic extracellular polymeric
matrix which may sequester RONS and attenuate plasma cidal efficacy and maintains a 3D architecture supporting heterogeneous microenvironments that in turn
support multispecies microcolonies. Growth rate may be reduced due to nutrient and O2 limitations within the biofilm, leading to elevated tolerance and persister
formation. Quorum sensing, leading to alterations in microbial physiology may also affect microbial tolerance to plasma-derived RONS. Finally, RONS-mediated
dispersal of microbes from the biofilm may reverse plasma tolerance. Adapted from [7,87]. Abbreviations: eDNA, extracellular DNA; RONS, reactive oxygen and
nitrogen species.

Trends in Biotechnology, June 2018, Vol. 36, No. 6 631

 Plasma source

Electrode
Dielectric
barrier

Aer glow
Plasma plasma jet
discharge

Biofilm

(A) (B)
Electrode

Figure 2. Schematics of (A) a DBD CAP Source and (B) a CAP Jet Source for Biofilm Eradication and Control. In A (direct plasma exposure), bacterial
biofilm cells are exposed to plasma reactive species, charged particles, high electric fields/current and UV. In B (indirect plasma exposure), bacterial biofilm cells are
exposed primarily to reactive oxygen and nitrogen species generated in the plasma afterglow and not the electric fields/current or charged particles. Adapted from
[12,26]. Abbreviations: CAP, cold atmospheric plasma; DBD, dielectric barrier discharge.

hypoxic and anoxic conditions, where NO3 is reduced to yield, for example, NO, N2O and N2
gases [40]. In vivo, hypoxia may be driven by polymorphonuclear (PMN) leucocyte response to
a pathogen, where significant O2 consumption occurs during the respiratory or oxidative burst
(production of ROS) response and in the production of NO [40–42]. This consumption of O2 can
lead to hypoxic conditions in the cystic fibrosis (CF) lung, where the PMN-driven reduction in O2
leads to reduced growth rates and increased tolerance to antibiotics [40]. Chronic exposure to
PMN-derived ROS, superoxide and NO in vivo could predispose certain pathogens to display
inherent tolerance towards CAP treatment. This may explain observations of significantly
elevated tolerance to CAP treatment in clinical isolates from chronic infection, when compared
with laboratory or type strains [43]. However, the observed elevated tolerance to plasma
exposure in biofilms is likely to be multifactorial. Here, nonthermal plasma could have a
significant advantage over conventional antibiotics or biocides, since the chemical diversity
and flexibility created by nonthermal plasmas may be refined and tailored for such applications,
creating a dynamic and responsive therapeutic approach.

Biofilm Matrix Interaction with Plasma-Derived RONS

Biofilm matrix components such as eDNA and polysaccharides may also attenuate the
antimicrobial activity of CAP exposure via interaction and sequestration of plasma-derived
RONS. Polysaccharides, such as alginate, have been shown to interact with RONS and
therefore may contribute to the attenuation of the biocidal activity of CAP, similar to the effect
exogenous organic matter on biocidal disinfectant activity when evaluated under ‘dirty’

632 Trends in Biotechnology, June 2018, Vol. 36, No. 6

conditions in standard efficacy tests. Alginate, an anionic polysaccharide, is a major component
of mucoid biofilms of P. aeruginosa (and other pseudomonads), where it performs a range of
vital functions including cell–cell adhesion, maintenance of biofilm fluidity and architecture, and
resistance to desiccation [44].

Importantly, alginate can also scavenge free radicals, primarily ROS, released by neutrophils
and macrophages as part of the respiratory or oxidative burst in response to pathogens [45].
Furthermore, alginate binds, limits diffusion of, and inhibits killing by cationic antimicrobial
agents including antimicrobial peptides [46] and antibiotics such as tobramycin [47,48] and
other aminoglycosides [49], leading to reduced EPS diffusion. The important role played by the
biofilm EPS matrix in conferring elevated antimicrobial tolerance, both by providing a physio-
logical shield preventing efficient ingress of toxic compounds such as antibiotics and biocidal
agents, and by maintaining hydration, architecture and microenvironments within the biofilm, is
increasingly recognized [2,3].

Another major component of many biofilms, including those of human pathogens such as
Staphylococcus aureus and P. aeruginosa, is eDNA [3,4]. eDNA forms an integral component
of the EPS, where it contributes to biofilm structure and architecture and to antimicrobial
tolerance towards the aminoglycosides and cationic antimicrobial peptides. It has also been
shown to induce tolerance to aminoglycoside antibiotics via acidification of the biofilm matrix
[50]. Interestingly, exposure of Staphylococcus epidermidis biofilms to low (subinhibitory)
doses of vancomycin increased the concentration of eDNA in those biofilms, impeding the
penetration of vancomycin through the biofilm and increasing antibiotic tolerance [51]. Numer-
ous studies have examined the effect of CAP on cellular DNA (from various biological sources).
In each case, rapid degradation of DNA has been demonstrated, whereby DNA undergoes
single- and double-strand breakage, the latter being regarded as a lethal event, with production
of ROS recognized as an important causative factor [52–54]. The rapid accumulation of single-
and double-strand breaks in DNA results in rapid fragmentation of the DNA into smaller
oligonucleotides [54]. CAP-activated phosphate-buffered saline also gives rise to oxidative
DNA damage and biocidal effects in Escherichia coli [55]. Clearly, eDNA sequesters a propor-
tion of the reactive species during these events, however, rapid degradation of eDNA in the
biofilm matrix following CAP exposure could increase the penetration of plasma-active species
into the biofilm matrix, leading to increased rates of inactivation. Used alongside conventional
antibiotics and biocides, CAP-mediated increased penetration could lead to synergistic effects.
The effect of plasma-mediated eDNA degradation on biofilm viscoelasticity, architecture, and
antimicrobial penetration warrants further investigation.

A recent study examined the susceptibility of biofilms of clinical isolates of the CF-associated
pathogen Burkholderia cepacia [43]. Clinical isolates that produced the greatest biomass (EPS)
were least sensitive to the bactericidal effects of CAP. Strains producing greatest biomass in
vitro also exhibited the highest catalase activity, suggesting the enzymatic inactivation of CAP-
derived ROS may have contributed to survival [43]. In a supporting study, catalase production
by P. aeruginosa prevented penetration of H2O2, and protected aggregated bacteria within the
biofilm [56]. Such highly CAP-tolerant biofilms often exhibit biphasic time–kill curves, with a
marked time-dependent tailing in the rate of killing (after an initial rapid decline in bioburden) and
often no complete eradication of the population [43,57]. However, the exact mechanisms by
which RONS either stimulate or circumvent bacterial tolerance remain poorly understood.
Despite this, evidence to support CAP-mediated tolerance and development of a persister
phenotype, is emerging.

Trends in Biotechnology, June 2018, Vol. 36, No. 6 633

Plasma-Induced Persistence and Tolerance
Persister cells (which may be within biofilms or in the planktonic phase) are transient phenotypic
variants in a microbial population, which exhibit metabolic inactivity, dormancy, and transient,
high-level tolerance to stress, such as antimicrobial insult and starvation [58], and present
additional challenges in effective biofilm control. Unlike resistant mutants, persistence does not
occur by mutation, but rather by phenotypic variation and is thus nonheritable. This phenotypic
variation results in a metabolically inactive, quiescent state whereby the cell neither grows nor
dies in when exposed to a lethal stress [59]. Previously, the two most well-characterized states
of microbial dormancy, persister cells and viable but nonculturable (VBNC) cells, which
allow bacteria to tolerate and withstand environmental stress, were believed to coexist
stochastically in microbial populations [60]. This work supported the ‘dormancy continuum
hypothesis’ whereby the VBNC and persister cells utilize similar mechanisms of dormancy but
occupy different physiological positions in the dormancy range [61]. The key difference in these
studies was the speed of resuscitation of a culturable phenotype upon the stressor being
removed [60]. However, recent work by Kim and co-workers has suggested that VBNC cells
and persister cells are in fact the same dormant phenotype [62].

CAP exposure can induce the VBNC phenotype in bacterial populations exposed to nonther-
mal plasma [57,63–65]. More recently, persister cell formation following plasma exposure in P.
aeruginosa has been described, with the redox-active molecule phenazine reported to play a
role in the bacterial response. The results of this study clearly indicate the central role, played by
oxidative stress in CAP-mediated killing and phenotypic alteration towards persistence [65].
Therefore, the ability to induce oxidative stress in bacterial populations, specifically in biofilms,
by low level, sublethal CAP exposure needs to be carefully considered since it may prove a
driver of cellular dormancy, mutation, and resistance, and incomplete eradication of the target
microbial population. However, CAP treatment regimens, which rely on repeated application
may, by altering the CAP/RONS profile, obviate such issues. Indeed, the specific CAP-derived
stimuli which trigger these phenotypic responses warrants further investigation, as do the CAP
generated reactive species which could potentially resuscitate persister or VBNC cells in the
heterogeneous environment of the biofilm and return them to a metabolically active, antimi-
crobial sensitive phenotype.

The ability of repeated CAP exposures to lead to plasma resistance has been investigated
recently [66]. In this work, MRSA and Enterococcus mundtii (Gram positive) and E. coli (Gram
negative) exhibited no primary (natural) or acquired resistance to CAP after repeated exposure.
This is perhaps unsurprising given the potential range of cellular targets of plasma-derived
reactive species [67] and the ability of different reactive species to target discreet cellular death
pathways [68]. However, these studies have been conducted in planktonic cultures, and do not
take into account the effect of the privileged environment and heterogeneity of the biofilm.

NO-Induced Biofilm Dispersal

NO has been proposed to be a highly conserved regulator of dispersal in eukaryotes [69]. In the
past decade, NO has been identified as a dispersal signal in a number of diverse bacteria,
including P. aeruginosa, S. epidermidis, Serratia marcescens, Vibrio cholera and Fusobacte-
rium nucleatum [70,71], modulating dispersal and antibiotic tolerance. A further study demon-
strated that nitrite-induced stress in S. aureus resulted in impairment of both polysaccharide
intracellular adhesion (PIA) synthesis and biofilm formation. Nitrite induced stress led to
upregulation of genes associated with oxidative and nitrosative stress (including genes for
DNA repair, iron homeostasis, and detoxification of ROS). Nitrite addition also eradicated
preformed biofilms of S. aureus and repressed biofilm formation in S. epidermidis. Interestingly,

634 Trends in Biotechnology, June 2018, Vol. 36, No. 6

nitrite-induced biofilm effects are repressed by addition of NO scavengers (carboxy-PTIO and
bovine hemoglobin), suggesting an important role for NO in these processes [72]. Furthermore,
NO has been reported to potentiate killing of B. cepacia by ROS [73]. Recently, a randomized
UK clinical trial of inhaled low-dose (10 ppm) NO as an adjunctive therapy to treat chronic
P. aeruginosa infection in CF patients demonstrated that NO led to disruption of biofilms in CF
sputum, decreased tolerance to tobramycin and tobramycin/ceftazidime, and significant
reduction in P. aeruginosa biofilms aggregates in vivo [74].

NO generation in nonthermal plasma jets has been described previously [75,76], where it was
found to be the dominant long-lived species in high concentrations (concentration was
dependent on jet configuration and operating conditions) and positively correlated with micro-
bial inactivation rates [76]. Plasma-derived NO and RNS are therefore likely to participate in
both direct killing in the biofilm, as well dispersal or activation of cell stress responses when
exposed to nonlethal concentrations of NO. For example, RNS (particularly NO) have been
shown to activate the SOS DNA repair response in E. coli [77]. The effect of sublethal exposure
to nonthermal plasma-derived RONS in biofilms during clinical application of this technology
contributes to understanding the complex mechanisms of plasma interaction with biofilm
communities and the bactericidal/survival mechanism at play.

CAP Interference with Bacterial Quorum Sensing

Quorum sensing (QS) is a population-density-dependent transcriptional regulation mecha-
nism (bacterial cell-to-cell signaling mechanisms reliant on the production and accumulation of
self-produced extracellular chemical signals, called autoinducers), which permits bacterial
populations to respond collectively to external stimuli or stress, and coordinate group behav-
iors, such as cellular differentiation, production of specific metabolites and biofilm formation
[78–80]. In such systems, as bacterial cell number increases, the accumulation of the bacterial
signaling molecule also increases until a threshold concentration is reached, whereupon gene
transcription or repression occurs. The best characterized of these signaling networks involves
the broad class of N-acyl homoserine lactone signaling molecules. Based on observations that
QS-deficient mutants are attenuated for virulence and antimicrobial tolerance [81], QS inhibition
by exogenous inhibitors has been demonstrated to control (for example) biofilm formation [82]
and modulation has been proposed to control bacterial virulence and infection. For an excellent
recent review, the reader is directed to [80].

QS is an attractive therapeutic target in biofilm control, since QS inhibitors or agents which

otherwise attenuate cell-to-cell signaling via modification of the autoinducer molecule
(e.g. acylases or lactonases), have the potential to reduce virulence factor production, increase
antimicrobial susceptibility, and potentially reduce biofilm formation without imposing selective
pressure on the target pathogen, which could ultimately lead to emergence of resistance.
Relevant to development of CAP biofilm control applications, QS regulates expression of both
catalase and superoxide dismutase in P. aeruginosa and is necessary for biofilm tolerance to
H2O2 [83]. The ability of CAP exposures to chemically modify and inactivate N-acyl homoserine
lactone QS signaling molecules, to inactivate QS-regulated virulence factors elastase (lasB) and
pyocyanin [84], and to inhibit QS-regulated production of virulence factors has recently been
demonstrated [85]. In addition to inactivation of other proteolytic enzymes in a time-dependent
fashion [67,68], the combined effect of reduction in biofilm viability, the destruction of enzymatic
activity of virulence factors and modification/inactivation of QS molecules, could facilitate
healing and infection resolution in scenarios such as chronic wound biofilms. The application
of CAP may also be described as an antivirulence intervention in the context of chronic biofilm
infections that may require multiple exposures.

Trends in Biotechnology, June 2018, Vol. 36, No. 6 635

Concluding Remarks Outstanding Questions
A number of significant gaps in our current understanding of mechanism of action, relative What are the most important antibio-
importance of individual plasma-derived reactive species, and in understanding the long-term film species produced by CAP? Are all
CAP RONS beneficial in controlling
role of CAP in management of bacterial biofilm infections remain to be fully addressed (see biofilms?
Outstanding Questions). The development of devices for biofilm control is an area of active
research. However, considering the multiplicity of biomedical, environmental, and agricultural Can tolerance to CAP be overcome by
niches where biofilms thrive, it is unlikely that a single device will suit all applications. A key development of a responsive and flexi-
ble profile of plasma-generated reac-
challenge for in vivo treatment of patients and tissues for wound healing or drug activation
tive species, which may be optimized
purposes relates to local temperature increases and toxicity to nontarget surrounding cells. for maximum efficacy according to
Ensuring complete inactivation of cells within biofilms prior to removal or matrix degradation is bacterial species and stage of
critical, to mitigate the potential for dislodgement to an alternative location and emergence of infection?
infection or contamination at a different site. The cytotoxic and mutagenic potential of cold
Does CAP exposure induce a persister
plasma requires further evaluation specific to device and application. There is a small range of
phenotype, particularly in biofilms
approved commercially available systems for clinical use, and while cold plasma demonstrates where EPS-embedded microcolonies
enormous therapeutic potential, ensuring the biological safety will promote clinical adoption may be exposed to repeated subinhib-
and broader regulatory approval. itory concentrations of ROS and RNS?

Can repeated subinhibitory CAP expo-

As a therapeutic approach to the management of microbial biofilms in clinical practice,
sures ultimately lead to the emergence
nonthermal plasmas may have significant advantages over conventional antimicrobial of tolerance or resistance to CAP-gen-
approaches. Principally, the ability to tailor the plasma-generated reactive species to target erated reactive species?
the multiple targets within the biofilm, discussed herein, is particularly attractive. Recently, the
tailored generation of aqueous ROS in plasma-activated water has been demonstrated with AC How can omics technologies best be
applied to glean important information
spark and glow discharge sources in direct contact with water [86]. However, not all plasma- regarding the response of biofilm bac-
generated RONS may prove beneficial in biofilm control. Challenges remain in understanding teria to specific RONS, to enable opti-
the fundamental plasma–biofilm interactions that may drive tolerance and persistence in mization of CAP for biofilm
microbial consortia. This will require fundamental studies in both plasma physics and diagnostic applications?

evaluation of the diverse range of plasma sources currently used, and fundamental biofilm
What parameters should be consid-
investigations including mass transfer of reactive species into the biofilm matrix, matrix inter- ered for the development of MICAPE?
actions with plasma-derived reactive species, and their effects on populations surviving within
the biofilm, where tolerance and ultimately resistance may emerge.

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