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Nat Rev Microbiol. Author manuscript; available in PMC 2018 June 01.
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Published in final edited form as:

Nat Rev Microbiol. 2017 December ; 15(12): 740–755. doi:10.1038/nrmicro.2017.99.
Targeting microbial biofilms: current and prospective

therapeutic strategies
Hyun Kooa,*, Raymond N Allanb,c,%, Robert P Howlind,e,%, Luanne Hall-Stoodleye,f,$, and
Paul Stoodleyf,g,h,$,*
aBiofilmResearch Labs, Levy Center for Oral Health, Department of Orthodontics and Divisions
of Pediatric Dentistry & Community Oral Health, School of Dental Medicine, University of
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Pennsylvania, PA, USA.

bClinicaland Experimental Sciences, Faculty of Medicine and Institute for Life Sciences,
University of Southampton, Southampton, UK.
cSouthampton NIHR Wellcome Trust Clinical Research Facility, University Hospital Southampton
NHS Foundation Trust, Southampton, UK.
dCentre for Biological Sciences, University of Southampton, Southampton, UK.
eSouthampton NIHR Respiratory Biomedical Research Unit, University Hospital Southampton
NHS Foundation Trust, Southampton, UK.
fDepartment of Microbial Infection and Immunity, Centre for Microbial Interface Biology, The Ohio
State University, Columbus, Ohio, USA.
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gDepts. Orthopaedics and Microbiology, The Ohio State University, Columbus, Ohio, USA.
hNational Center for Advanced Tribology at Southampton (nCATS), Faculty of Engineering and
the Environment, University of Southampton, UK.
Abstract
Biofilm formation is a key virulence factor for a wide range of microorganisms that cause chronic
infections. The multifactorial nature of biofilm development and drug tolerance imposes great
challenges for the use of conventional antimicrobials, and indicates the need for multi-targeted or
combinatorial therapies. In this review, we focus on current therapeutic strategies and those that
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*
Corresponding authors: Koo, Hyun ([email protected]): University of Pennsylvania, School of Dental Medicine, 240 South 40th
Street, Levy Bldg. Rm 417, Philadelphia, PA 19104, USA. Stoodley, Paul ([email protected]): The Ohio State University,
Centre for Microbial Interface Biology, 716 BRT, 640 W 12th Ave., Columbus, OH 43210. USA.
%Equal contribution
$Equal contribution
Author contributions
Hyun Koo. Contribution of writing and editing to all sections and figures. Preparation of Fig. 4.
Raymond N Allan. Contribution of writing and editing to all sections and figures. Preparation of first draft of Fig. 2.
Robert P Howlin. Contribution of writing and editing to all sections and figures. Preparation of first draft of Fig. 3.
Luanne Hall-Stoodley. Conceptualized the original outline of the article. Contribution of writing and editing to all sections and figures.
Paul Stoodley. Conceptualized the original outline of the article. Preparation of first draft of Fig. 1. Contribution of writing and editing
to all sections and figures.
Competing Interests Statement
The authors declare competing interests.
Koo et al. Page 2
are under development that target vital structural and functional traits of microbial biofilms and
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drug tolerance mechanisms, including the extracellular matrix and dormant cells. We emphasize
strategies that are supported by in vivo or ex vivo studies, highlight emerging biofilm-targeting
technologies, and provide a rationale for multi-targeted therapies that are aimed at disrupting the
complex biofilm microenvironment.
Initially reported as an arcane behaviour of bacterial populations, microbial biofilm

formation is now recognized as a principle virulence factor in many localised chronic
infections. Biofilm infections commonly recur after long periods of clinical quiescence. This
is not primarily due to genetic resistance that arises by mutation, although the increased
microbial cell density may favour transfer of resistance genes. Rather microorganisms that
reside in biofilms may develop tolerance to traditional antibiotics or antimicrobial agents
through metabolic dormancy or molecular persistence programmes. Moreover, the important
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role of the extracellular matrix in conferring antimicrobial tolerance to biofilms is being

recognized1. Advances in multi-omic and imaging technologies have also revealed the
remarkable complexity and spatial organization of polymicrobial biofilm infections2.
Accordingly, our increased understanding of biofilms is rapidly changing the strategies used
to treat these challenging infections (Fig. 1). Nonetheless, the control of biofilm formation
and treating existing biofilms remains tenuous with few new therapeutic options currently
available clinically.
Biofilm infections are not easily amenable to existing antimicrobial treatment or ‘single
magic bullet’ approaches, because biofilm recalcitrance is a consequence of complex
physical and biological properties with multiple microbial genetic and molecular factors,
and also frequently involve multi-species interactions. A diverse range of microorganisms
(Gram-positive and Gram-negative, motile and non-motile, aerobic, anaerobic and
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facultative bacteria, and fungi) form biofilms, which share many common features (Box 1).
Although the ‘universal’ role of cell signalling in biofilm formation was revealed 20 years
ago, signalling-based therapeutics have yet to be introduced for the clinical management of
biofilm infections owing to the complexity in cell signalling networks. Similarly, the
emergence of materials science, the development of surface modifications that incorporate
technologies that target adhesion, as well as biomimicry or surface textures and chemistries
from plants and animals3 were promising approaches to prevent microbial adherence and
subsequent biofilm formation. Although many studies show statistical significant reductions
in biofilm or alterations in biofilm structures in the laboratory, few were tested or validated
using in vivo or human cell models to see if they translated to clinical significance. Many
studies only report early time points, fail to use clinically relevant treatment regimens or do
not consider the presence of molecularly complex host fluids or host cells at the site of
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biofilm infections. More recent approaches include targeting the extracellular polymeric
substance (EPS) matrix. However, the variability in the composition of the EPS matrix and
the interactions among the various components4 add new levels of complexity and provide
challenges for the development of EPS-targeting therapeutics5.
Several excellent reviews discuss how microorganisms develop pathogenic biofilms and
their protective mechanisms against antibiotics, antimicrobial agents and host innate
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immunity 1,6,7. This Review focusses on the challenges facing the development of biofilm-
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specific therapeutic strategies, how new insights into the chemical composition and structure
of the EPS matrix, active biofilm dispersal pathways, and recognition of the role of dormant
persister cells or slow-growing subpopulations in conferring antibiotic tolerance are being
exploited to target biofilm infections. We also review developing technologies that promise
to enhance the efficacy of current modalities or provide novel biofilm-targeting effects,
including challenges to ensure biocompatibility and therapeutic efficacy, which are both
critical for clinical translatability. Where possible we focus on technologies that have shown
efficacy in preclinical trials, or robust animal or human cell infection models. However, as
not all potential biofilm-targeting therapeutic strategies can be discussed in detail, we
provide a comprehensive list of current and prospective biofilm-targeting strategies, their
developmental stage and a brief list of advantages and disadvantages in Supplemental Table
S1.
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Finally, we contend that treating biofilm infections requires combination therapies or those
that target more than one component of the complex biofilm microenvironment, similar to
tumouregenesis8. Importantly, biofilm infections reflect an interplay between the host and
opportunistic pathogens, often within a complex microbiota. Polymicrobial biofilms pose an
additional challenge, requiring antimicrobials that are effective against all pathogenic
microorganisms in the biofilm and limiting the efficacy of species-specific biofilm-targeting
strategies. All of these challenges contribute to why so few therapies have yet to be
translated clinically.
Current therapeutic approaches

Many biofilm management strategies being devised in the clinic and used by surgeons, are
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largely based on an approach from cancer treatment (Box 2): early and aggressive irrigation
and debridement for physically removal and local delivery of high and sustained
antimicrobial chemotherapy9. Given the devastating consequences if a biofilm infection
persists, surgeons are undertaking earlier and more aggressive treatment, including revisiting
‘old’ last-resort antibiotics such as colistin10. Another established approach used for
intravenous catheter-related infections is lock therapy11. Following a decision to treat rather
than remove certain types of catheters, the potential to leave biofilms intact (but containing
dead cells) includes the potential to promote colonization by other microorganisms. This
illustrates a crucial point regarding biofilms: killing does not necessarily eradicate the
biofilm. Therefore the challenge of using antimicrobial agents, which may kill
microorganisms but leave behind other biofilm components, must be addressed.
Since understanding the mechanisms of biofilm formation derives primarily from how they
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form on solid surfaces, most clinical trials testing biofilm-targeting approaches or FDA-
approved therapeutics have focussed on indwelling medical devices. Current biofilm-
targeting technologies can be divided into two groups: physical-mechanical approaches (for
example, high velocity spray and jet irrigators) that are aimed at biofilm disruption and
removal; and surface-coating or eluting substrates, which can be impregnated with
antibiotics and/or antimicrobials (for example, acrylic beads with absorbable antibiotic-
loaded bone cement to prevent orthopaedic infection12) for biofilm prevention; where higher
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localised antibiotic concentrations can be achieved for longer periods by in situ antibiotic
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delivery compared to systemic administration. Several antimicrobial metal or inorganic

coatings have also reached clinical application to prevent biofilm formation13. These include
silver coating in endotracheal tubes, catheters, megaprostheses, wound dressings and copper
alloys in hospital surfaces (supplemental Table S1). With respect to treating prexisiting
biofilms, laboratory studies show that statistically significant reductions in biofilm viability
can require extended incubation periods with high antibiotic concentrations. In situ release
offers an important approach to achieve such high concentrations over long treatment
periods 14,15. Although antibiotic-impregnated beads in bone cements or dental restorative
materials were used before biofilm formation was recognized as a distinct etiological factor,
they represent a class of technologies that are now being re-examined to gain better
understanding of their effect in controlling biofilm infections.16
Mechanical disruption using water sprays and jets have been developed and used for
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pathogenic biofilm removal and for irrigation, including debridement of surgical site
infections to remove necrotic tissue, exudates or dental biofilms. High-speed imaging has
provided important information on fluid-biofilm-surface interactions and show that although
a statistically significant amount of biofilm is removed from the area, the biofilm becomes
fluidized and spreads across the surface17. The ability of biofilms to become fluidized
probably explains the tenacity of bacteria on surfaces after pulsed lavage18 and may
contribute to the low success rate of irrigation and debridement alone in treating
periprosthetic infections. An advantage of water-based jets is that antimicrobial agents can
be readily added so that the fluid doubles as a delivery device as well as creating mechanical
forces acting on the biofilm. However, despite advances in biofilm specific-clinical
therapies, particularly for indwelling devices, most approaches still entail conventional
antibiotic-based therapy or topical broad-spectrum antimicrobials.
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EPS-targeting strategies
The composition of the EPS matrix is temporally and structurally variable depending on the
type of microorganism, local mechanical shear forces, substrate availability and the host
environment. The EPS matrix promotes microbial adherence to a surface, cell-cell adhesion
and aggregation19, and functions as a 3D scaffold that provides cohesiveness, mechanical
stability and protection against host effectors and antimicrobial therapies. In addition, the
EPS matrix can dynamically modulate chemical and nutrient gradients, and delineate
pathogenic environments (such as acidic pH and hypoxia), which contribute to key virulence
attributes, including recalcitrance1,4. Thus, targeting the EPS may be an effective strategy to
remove biofilm, disaggregate bacteria and disrupt the pathogenic environment20. Targeting
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can be achieved by: inhibiting EPS production, binding EPS adhesins on the microbial
surfaces to block adhesion, or by degrading EPS in established biofilms (Fig 2).
Disrupting EPS synthesis and secretion, and binding of EPS adhesins

Several extracellular and intracellular signalling networks as well as non-signalling
mechanisms that promote the production of EPS have been identified. In general, cyclic-di-
GMP and cyclic-di-AMP21 control various EPS-producing exoenzymes, polysaccharides
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and adhesins that are potential candidates that can be targeted to inhibit or disrupt EPS22,23.
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These nucleotide-signalling molecules regulate glucan-producing exoenzymes (for example,

glucosyltransferase) in Gram-positive Streptococcus mutans as well as the aggregative
exopolysaccharides Psl and Pel in Gram-negative P. aeruginosa. Several potential small
inhibitors of di-guanylate or di-adenylyl cyclase have been identified through library
screening or in silico drug discovery combined with bioactivity assessment using in vitro
biofilm models,24,25 although their efficacy against biofilms awaits further in vivo
validation.
The inhibition of EPS glucan synthesis by glucosyltransferase using small-molecule

inhibitors reduced the accumulation of pathogenic biofilms on teeth, and supressed the onset
of oral diseases in vivo without disturbing the resident microbiota.26,27 These small-
molecule inhibitors alone are not superior than current chemical modalities for oral biofilm
control (chlorhexidine) or tooth decay prevention (fluoride), however, when used in
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combination, EPS inhibitors can greatly enhance their therapeutic effects26. Inhibitors of
adhesin production and adhesin-binding antibodies or peptides have also been developed to
disrupt bacterial binding to host surfaces. Small molecules (for example, peptides or
mannosides) that target host- EPS matrix interactions have shown efficacy in prevention and
treatment of both bacterial and fungal biofilm infections in vivo.28,29 Mannosides that target
the bacterial adhesin FimH (alone or combined with trimethoprim-sulfamethoxazole)
prevented catheter-associated urinary tract infection (UTI) in mice by reducing Escherichia
coli colonization 2-log, and treated chronic cystitis by reducing the E. coli population 3-
log28,30,31. A recent study has also attempted to address the low half-life and bioavailability
of O-mannosides by generating C-mannosides, which have increased metabolic stability and
in vivo efficacy. Prophylactic treatment with this compound reduced the E. coli burden 2-log
and treatment of chronic infection resulted in a 4-log reduction in a UTI mouse model32.
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Similarly, ring-fused 2-pyridones, which inhibit the biogenesis of curli and type-I pili,
reduced uropathogenic E. coli bladder colonisation more than 10-fold and the establishment
of intracellular bacterial communities in an in vivo mouse UTI model33. Several other
biomolecules that bind to EPS adhesins are discussed in detail elsewhere34.
Targeting EPS chemical composition and structure

Exopolysaccharide-degrading enzymes such as glucanohydrolases (dextranase and
mutanase) and dispersin B can disrupt the matrix of pathogenic oral biofilms, and glycoside
hydrolases have been used to degrade a mixed-species S. aureus and P. aeruginosa biofilm
grown in a mouse model of chronic wounds 35–37, although poor retention and enzymatic
stability (for example, susceptibility to proteolysis) may compromise efficacy in vivo36.
Nevertheless, a purified serine protease, Esp, from S. epidermidis inhibited S. aureus biofilm
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formation and eradicated pre-existing biofilms in vitro, while the susceptibility to the
antimicrobial β-defensin 2 was enhanced and S. aureus nasal colonization in humans was
reduced38. Another approach used endolysins (bacteriophage-encoded peptidoglycan
hydrolases), which enzymatically degraded the bacterial cell wall peptidoglycan 39.
Engineered peptidoglycan hydrolase constructs with distinct antimicrobial activities
degraded multiple unique bonds in the peptidoglycan structure specific to S. aureus40, and
increased killing and biofilm removal in animal models. Fusion proteins derived from
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multiple bacteriophage-encoded endolysins, each with unique actions, all added

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simultaneously may also reduce the risk of resistance, yet show sufficient specificity to avoid
targeting commensal strains. Glycoside hydrolases were recently shown to both disrupt pre-
existing P. aeruginosa biofilms and potentiate neutrophil mediated killing41.
DNases have also shown efficacy in disrupting biofilms42. Consistent with the role of eDNA
in the EPS matrix and in early biofilm development, DNase I is effective in disrupting early
biofilms in vitro and in vivo42,43. Notably, other biomolecules, including polysaccharides
and various proteins that are associated with eDNA contribute to biofilm structural integrity,
which may explain the efficacy of DNase I in treating nascent biofilms. Few studies have
used DNases to specifically target biofilms in vivo, however, it was shown to statistically
significantly decrease Gardnerella vaginalis colonization on vaginal mucosal epithelial cells
in a mouse model44. Therapeutic use of recombinant human DNase I (dornase alfa) degrades
neutrophil and microorganism-derived DNA in the sputum of patients with cystic fibrosis,
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thus reducing sputum viscosity45. An intervention study with dornase alfa in patients with
cystic fibrosis and early lung disease showed significantly improved lung function and lower
risk of exacerbation compared to placebo groups, with a potential decrease in the rate of
lung function decline in children46. A clinical trial investigating the efficacy of dornase alfa
for the treatment of chronic otitis media, at the time of tympanostomy tube insertion to
promote bacterial clearance from the middle ear combined with antibiotic drops, is under
evaluation47,48.
Matrix-degrading enzymes can help disperse bacteria in biofilms for more effective killing
when combined with antimicrobial agents. Targeting EPS can also disrupt the viscoelastic
properties to further weaken biofilm cohesiveness and enhance antimicrobial efficacy,
including host mediated antimicrobial responses. Recent studies showed that glucano-
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hydrolases, glycoside-hydrolases and DNases enhanced antimicrobial delivery and

potentiated killing by antibiotics or antimicrobial peptides when used in combination against
pre-formed biofilms in vitro49,50. Overall, EPS synthesis inhibitors or EPS-degrading
enzymes, which lack intrinsic antibacterial activity, seem to be a promising adjunctive
approach for biofilm control that could potentially enhance the killing efficacy of
antimicrobial agents and promote biofilm removal when co-administered.
EPS-targeted antibodies and nucleic acid-binding proteins

Vaccine approaches pose several challenges as a strategy to target biofilms , as vaccines are
specific to a microorganism and clinical isolates from biofilm infections show considerable
variability in genotype and/or the phenotypic expression of vaccine-targeted epitopes51. A
more effective approach may be to use antibodies targeted specific EPS components.
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Monoclonal antibodies against P. aeruginosa-derived EPS identified epitopes that bound to

the polysaccharide Psl, which is widely present in P. aeruginosa clinical isolates52. Psl was
shown to be a serotype-independent, antibody-accessible antigen, and anti-Psl antibodies
increased opsonophagocytic killing of P. aeruginosa, inhibited attachment to lung epithelial
cells in vitro, and showed prophylactic protection in multiple animal models of P. aeruginosa
infection. Additionally, vaccine-elicited antibodies to Enterococcus faecalis pilus tip (EbpA)
abrogated bacterial binding to fibrinogen and biofilm formation in a mouse model of
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catheter-associated urinary tract infections (CAUTI)53. Notably, EbpA did not mediate E.
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faecalis adhesion directly to the catheter material, but rather inhibited binding to fibrinogen
deposited on the catheter surface. The EbpA antibody response prevented EbpA-mediated
fibrinogen-dependent bacterial aggregation and biofilm formation on catheters. This
approach highlights why using a complex host-microorganism model can reveal additional
targets. In another approach, a multivalent vaccine exploiting both planktonic and biofilm-
expressed polypeptides from S. aureus showed increased efficacy in combination with
antibiotics compared to antibiotic treatment alone in a rabbit model of osteomyelitis54.
Nonetheless, targeting broadly conserved components in EPS is desirable. The DNABII

family of DNA-binding proteins have a key role in providing structural integrity to eDNA55.
The high binding affinity of integration host factor (IHF) has specifically been exploited to
target nucleoproteins in biofilms and been widely tested in animal models. Antibodies
against E. coli IHF are cross-reactive and bind to DNABII in multiple bacterial species,
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resulting in biofilm destabilisation and the release of individual bacteria. When combined
with antibiotic therapy, immunotherapy targeting DNABII has shown efficacy in vivo
against biofilms in numerous types of bacteria, including oral bacteria56, uropathogenic E.
coli57 and P. aeruginosa in a mouse lung infection model58. This approach has also shown
efficacy against MRSA biofilms compared with antibiotic treatment alone in mouse
models59,60. In a combinatorial approach without using antibiotics, DNABII antibodies were
combined with a vaccine strategy. A study with nontypeable H. influenzae (NTHi) in an
animal model of otitis media used IHF and recombinant soluble type IV pili (rsPilA) co-
administered with an adjuvant and delivered by transcutaneous immunization to achieve
early NTHi eradication and prevention of disease 61. This approach also resulted in the
disassembly of NTHi biofilms that were established prior to immunization, thus leading to
resolution of existing disease.
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Inducing biofilm dispersal

Biofilm dispersal has been shown to be a regulated process that involves the degradation of
the EPS matrix, and the triggering of this response has provided research strategies designed
to promote biofilm self-disassembly. These approaches, for the most part, assume that
dispersed bacteria have returned to an active state akin to their planktonic phenotype,
rendering them more susceptible to conventional antibiotics. Furthermore, liberated inactive
cells will also have lost a degree of protection conferred by their association with the biofilm
community and structural organization. Regardless of their dispersed state, it remains vitally
important in the clinical setting that dispersive or exogenous EPS-degrading agents are
administered alongside systemic antibiotics to avoid recolonization or bacteremia, and
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potentially septicaemia.
Targeting cyclic-di-GMP pathway

The intracellular secondary messenger nucleotide c-di-GMP has a key role in the biofilm
lifecycle of both Gram-positive and Gram-negative bacteria, whereby increased levels
promote biofilm formation and reduced levels disassembly62. The enzymes governing c-di-
GMP levels, diguanylate cyclases (synthesis) and phosphodiesterases (breakdown), possess
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GGDEF, EAL and HD-GYP domains that are found in numerous bacterial phyla. This
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signalling pathway therefore offers an attractive strategy to target multiple species, although
the complexity of c-di-GMP regulation makes it challenging to control63. However, few
studies that show biofilm dispersal have used relevant cell models in vitro or in vivo animal
models. One study used a P. aeruginosa construct containing an exogenous E. coli
phosphodiesterase. When expression was induced in vivo it resulted in reduced c-di-GMP
and dispersal of biofilms on silicone implants in a mouse foreign body infection model64.
Although, in principle, this study supports the potential use of such phosphodiesterases as a
strategy to modulate c-di-GMP and target biofilms, the authors noted limitations of their
findings, including an increased bacterial burden in the spleen. C-di-GMP is also a potent
stimulator of host immunity via interferon responses, and therefore it may be difficult to
attribute effects on biofilms specifically in vivo65.
A well-characterized approach to modulate c-di-GMP levels is though nitric oxide (NO).

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NO was first shown to regulate c-di-GMP levels and mediate biofilm dispersal in
Pseudomonas aeruginosa66 at low concentrations, and these results have since been
reproduced in several other bacterial species67. However, the use of gaseous NO or
spontaneous NO-donors presents clinical challenges owing to potential cytotoxicity from
systemic exposure, lack of specificity in targeting biofilm infections and cost. In addition, as
NO is labile, the optimal concentration to disperse biofilms is difficult to measure;. however
NO microelectrodes are highly sensitive and offer excellent spatial and temporal resolution
in tissues or body fluids. A proof-of-concept preclinical study using low-dose gaseous NO in
the pM to nM range, has recently shown to reduce the size of the P. aeruginosa biofilm
aggregate in sputum as a primary clinical outcome in a small number of patients with cystic
fibrosis68. Patients did not exhibit adverse effects to NO therapy. Although the biofilm
aggregate size was significantly decreased, NO did not reduce CFU as seen in another
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study69, perhaps because patients continued to receive antibiotic therapy throughout the
study period. However a Phase I clinical trial is ongoing to study the efficacy and safety of
NO in patients with cystic fibrosis70.
To address the cost of administering gaseous NO and potential systemic cytotoxicity issues,
cephalosporin-3´-diazeniumdiolates (C3Ds), composed of a stabilized diazeniumdiolate NO-
donor attached to the 3’-position of cephalosporin, have recently been developed to
selectively deliver NO to bacterial biofilms71. These pro-drug candidates are designed to
specifically release NO upon cleavage of the cephalosporin β-lactam ring via bacterial β-
lactamases and have been shown to be effective in dispersing in vitro P. aeruginosa
biofilms71. NTHi biofilms grown on primary ciliated epithelia also showed enhanced
sensitivity to the antibiotic azithromycin, reducing viability 2-log when a specific C3D,
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PYRRO-C3D, was used as an adjuvant; this response is possibly attributable to dispersal and
modulation of metabolic activity72. This effect was also demonstrated in a study using
primary epithelial cells from patients with primary ciliary dyskinesia (PCD), a disease that
compromises mucociliary clearance. Airway cells from patients with PCD showed increased
susceptibility to NTHi biofilm formation compared to epithelial cells from healthy
individuals, and PYRRO-C3D in combination with antibiotic significantly decreased NTHi
viability 2-log compared to antibiotic treatment alone73. Treatment of infected healthy
airway cells and infected airway cells from unhealthy individuals had no effect on
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transepithelial electrical resistance, which suggests that epithelial barrier function was
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unaffected. Although this alone is not a sufficient assessment of toxicity, the targeted release
of low NO concentrations (48 – 90 nM) should improve the safety of patients.
NO-donor instability is also an issue that is being addressed by developing nitroxides

(sterically hindered NO analogues) that exert biological responses via NO-mimetic
properties74. These molecules (carboxy-TEMPO, CTMIO, DCTEIO) elicited biofilm
dispersal in P. aeruginosa and E. coli similar to NO, with carboxy-TEMPO also reducing
tolerance to ciprofloxacin74,75. However, treatment with carboxy-TEMPO failed to disperse
MRSA biofilms, which indicates that this approach may be restricted to biofilms formed by
certain species. This highlights an ongoing concern for polymicrobial biofilms infections.
Other drugs that are currently under development include ciprofloxacin-nitroxide
conjugates, which similar to C3Ds, combine antibiotic activity with a donor compound76,
and fimbrolide-NO donor hybrids, which simultaneously target quorum sensing (QS) and
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NO pathways77.
Targeting quorum sensing

The role of QS systems in biofilm development and dispersal offers another intensely
studied strategy for the development of novel therapeutics. QS requires the binding of a
signalling molecule to a corresponding transcriptional regulator, which activates the
downstream transcription of select targets. As the production of many virulence
determinants in pathogenic bacteria requires cell-cell communication, QS inhibitors (QSI)
that target the AHL-QS system in Gram-negative bacteria or the QS systems in Gram-
positive bacteria have been extensively evaluated for efficacy on clinically relevant bacterial
biofilms using in vitro and in vivo models. For example, the QS autoinducer, AI-2, functions
as a chemorepellent in Helicobacter pylori by regulating the proportion and spatial
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organisation of biofilm cells78. Treatment of in vitro biofilms with exogenous AI-2 resulted
in both a reduction in the proportion of adherent cells and dispersal78. The autoinducing
peptide type I (AIP-I) also triggered dispersal in MRSA biofilms on titanium disks rendering
detached MRSA more susceptible to treatment with rifampicin and levofloxacin79.
Additionally, the use of a RNAIII-inhibiting peptide (RIP) resulted in a 7-log reduction in
MRSA compared to 5-log reductions observed with RIP-soaked Allevyn or teicoplanin
treatments alone in a mouse wound model80. A recent study used a high-throughput screen
to identify a benzamide-benzimidazole derivative, termed M64, that interferes with the
Pseudomonas quinolone signal (PQS) quorum sensing system, which regulates biofilm
formation and the production of virulence factors in P. aeruginosa81. Interestingly, M64
reduced both the virulence and persistence of the P. aeruginosa strain PA14 in a mouse
model of burn and lung infections when used alone, and it reduced the bacterial load further
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when used in combination with ciprofloxacin. M64 also did not exhibit cytotoxicity in
mouse macrophages and was shown to reduce the number of persister cells in the
population.
Although the increased efficacy of antibiotic treatment with QSI in vivo is promising,
reduced bacterial loads often depend on the strain and biofilm model82. Furthermore, QS
molecules can be washed away during biofilm initiation, whereas the EPS matrix can bind
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and sequester QS molecules and the effects may be limited to highly localized areas within
the biofilm structure1. Therefore, inhibitors need access and specific targeting to site of
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active QS-signalling. These factors in addition to the complexity in cell signalling networks,
make it a challenging therapeutic approach albeit such inhibitors can be used in combination
with other strategies.
Metabolic interference
The potential of exogenous amino acids in the treatment of biofilms has garnered
considerable interest, with specific amino acids having been shown to affect both biofilm
metabolism and development. L-arginine (L-Arg) functions as a substrate for alkali
production by arginolytic bacteria (for example, Streptococcus gordonii), which can
neutralize acids and modulate pH homeostasis within oral biofilms clinically83. Treatment of
polymicrobial biofilms comprising Streptococcus mutans, S. gordonii and Actinomyces
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naeslundii with L-Arg suppressed S. mutans growth and resulted in substantial reduction in
insoluble EPS and altered biofilm architecture84. In addition to pH modulatory effects83, L-
Arg also repressed genes involved in the production of insoluble EPS and bacteriocin in S.
mutans, while increasing hydrogen peroxide (used against S. mutans) production by S.
gordonii84 L-Arg reduced biomass and altered EPS architecture in S. gordonii biofilms85,
and also destabilized multispecies oral biofilms, thus reducing viability and increasing
susceptibility to cetylpyridinium chloride86. An alternative amino acid, L-methionine, was
also identified as a promising adjuvant for treating P. aeruginosa biofilms, triggering
disassembly and increasing sensitivity towards ciprofloxacin in a mouse model of chronic
pneumonia, and enhancing survival of infected mice87. This activity was attributed to up-
regulation of four different DNase genes and the subsequent degradation of eDNA in the
EPS matrix, although the exact pathways that regulate this response were not determined.
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Interestingly, L-Met seems to have been chosen for this study following screening of a
selection of D- amino acids and L- amino acids for their activity against P. aeruginosa
biofilms. Given the diversity in amino acid utilization between bacterial species it is unlikely
that a single amino acid would have a universal function, however, their importance, and that
of bacterial metabolism in general, should not be underestimated in the development of
future treatment strategies.
Another approach is based on evidence that iron metabolism is important in biofilm

formation in several pathogens88–91. Iron acquisition is crucial for pathogens to establish
infection, and epithelial cells containing the ΔF508 cystic fibrosis transmembrane
conductance regulator (CFTR) mutation showed that increased biofilm formation by
P.aeruginosa was linked to increased availability of iron92. Host defences normally actively
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sequester iron to limit the growth of infecting bacteria since iron is an essential nutrient.
However, Pseudomonas aeruginosa possesses multiple redundant iron receptor and uptake
systems such as production of the siderophore pyoverdin (an iron-chelating
molecule).However gallium, which is chemically similarto iron , is be taken up by bacteria
but not replace its functionality, thus inhibiting the iron-dependent pathways required for
cell growth and biofilm formation. This “Trojan horse” strategy interfered with P. aeruginosa
growth and iron metabolism, killed planktonic bacteria in an acute mouse pneumonia model
and reduced bacterial counts in established biofilms by 3 logs in a chronic biofilm lung
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infection model93. Gallium was administered via inhalation and importantly uptake was
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independent of the P. aeruginosa siderophore pyoverdin in vitro. However in vivo it was not
clear if gallium may have anti-inflammatory effects other than directly inhibiting biofilm
formation. Nonetheless, using iron chelators adjunctively with tobramycin reduced P.
aeruginosa in a co-culture model of human bronchial epithelial cells from a patient with
cystic fibrosis that carried the CFTR ΔF508 mutation, resulted in a 7-log reduction in viable
bacteria and also prevented biofilm formation on these cells94. More recently, the oxidation
state of iron was shown to be important95. This study examined mucus from the airways of
patients with cystic fibrosis and found that ferrous iron was the primary form of bioavailable
iron, which also correlated with the severity of cystic fibrosis lung disease, whereas ferric
iron did not. This study highlights the importance of directly investigating the phenotypic
state of bacteria in situ in human infections and its potential translational relevance in
informing new therapeutic approaches.
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Targeting dormant cells in biofilms

Targeting pathways to induce processes such as dispersal requires that cells are
metabolically active. However, available evidence also shows that dormant cells or persisters
residing within biofilms have a key role for drug tolerance (Box 3). It is therefore attractive
to consider antimicrobial approaches that physically or chemically disrupt cells rather than
interfering with cellular processes. Non-discriminating oxidizing agents such as
hypochlorite and hydrogen peroxide have been used as irrigants in wound96 and endodontic
debridement97. However studies reveal that even strong oxidizers such as sodium
hypochlorite fail to eradicate biofilms98 probably because long-term exposure is not possible
due to cytotoxicity concerns. Broad-spectrum cationic biguanides such as chlorhexidine or
quaternary ammonium adhere to cell walls and disrupts cell membranes. However,
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penetration was limited over the expected timescales used in ex-vivo dental biofilms99 with
longer term exposure increasing cytotoxicity, thus making this approach clinically
impractical.
Other exploratory avenues include antibiotics that are used for the treatment of infections
caused by slow-growing bacteria. Rifampin, used to treat staphylococcal orthopaedic-
implant infections, raises concern about the development of rifampin resistance. However,
used in combination with other antibiotics, rifampin and fosfomycin enhanced efficacy in
treating foreign body MRSA biofilm infections in vivo100. Likewise, disrupting a cellular
target in dormant cells can kill persisters. The acyldepsipeptide antibiotic (ADEP4) can
activate the ClpP protease in dormant persister cells in Gram-positive bacteria so the cells
effectively ‘digest’ themselves. Although it is an elegant concept to endogenously activating
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cytoplasmic enzymes for proteolytic degradation in biofilms, it should be noted that ClpP is
not an essential enzyme and ClpP-null mutants are not affected by ADEP4. To address this,
treatment with both ADEP4 and rifampin showed good efficacy in a chronic biofilm mouse
deep abscess-like infection model101 using various S. aureus species. However, this study
illustrates that careful consideration needs to be given to antibiotic pairings. Particularly in
this case since rifampin resistance occurs at high frequency and so it is usually combined
with other active antimicrobials. In the case of ClpP-null mutants ADEP4 would be
ineffective and rifampin would in effect be acting as a monotherapy
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Antimicrobial peptides (AMP) represent another approach in treating biofilms independent

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of the presence of microbial activity. An important advantage of AMPs is that they are
widely conserved and therefore attractive as broad-acting antimicrobial agents that may be
useful against both bacterial and fungal biofilms102,103. Conversely, species-specific
targeting is also possible with synthetic AMPs that consist of dual functionally independent
moieties (a broad-spectrum AMP with a killing moiety, and a species-specific binding
peptide with target specificity). This approach may remove specific pathogens such as S.
mutans from oral multispecies biofilm communities to promote a ‘healthy-like microbiome’
as was shown in vitro104. Another advantage is that the pore-forming activity of an AMP
targets respiring cells as well as persister and dormant populations, which might reduce the
potential for bacteria to develop AMP resistance. Therefore, AMPs have potential as
therapeutics to target biofilms. Synthetic peptides that modify specific AMP sequences were
designed that showed both inhibitory activity and, when used together with antibiotics,
enhanced killing of P. aeruginosa biofilms in invertebrate infection models105. Specific
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peptides also triggered the degradation of ppGpp, preventing the accumulation of this
secondary messenger and abrogating biofilm formation of several G-positive and G-negative
pathogens103. However, more pre-clinical efficacy studies are required as AMPs can bind to
EPS matrix components and to other host molecules, which reduces their effectiveness, and
microbial proteases may further diminish AMP potency106. Additionally, the high cost of
AMPs synthesis is a barrier for clinical development and commercialization, although using
chloroplast-based technology for large-scale production in automated greenhouses may
mitigate costs50. Nevertheless, AMPs can be immobilized onto solid surfaces to enhance
efficacy or specificity. This was particularly effective as a polymer-based approach on
catheters, as AMP coatings greatly reduced P. aeruginosa adhesion and infection over 7 days
in a mouse UTI model107. Furthermore, structurally nanoengineered AMP polymers
exhibited potent killing activity against several Gram-negative, colistin-resistant and MDR
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pathogens, and they exhibited low toxicity and efficacy in an animal model of Acinetobacter
baumannii infection108. The recent completion of two Phase II clinical studies of brilacidin
(a membrane-acting AMP mimetic) as an intravenous agent for skin infections demonstrate
the feasibility of AMPs for systemic therapeutics109.
AMPs can also enhance conventional antimicrobial activity, and the combination with
strategies that target the EPS matrix may further increase both the access and permeabilizing
properties of AMPs once in the biofilm50,103. Although targeting tolerant cells using AMPs
is a promising approach, reaching the target cells that are embedded within a biofilm either
topically or systemically and for the compound to be active across a spatially and chemically
heterogeneous microenvironment remain important challenges in vivo. The stability and
durability of AMP coatings within the body is also an issue that needs to be further
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addressed, particularly where wear might be expected due to shear caused by moving tissues
and fluids as well as proteolytic degradation.
The promise of new technologies

While our understanding of biofilm microenvironments is evolving, technological advances
have provided unprecedented avenues to develop multi-targeted therapeutic approaches that
prevent and disrupt biofilms or enhance drug efficacy (Fig. 3). Nano- and chemical
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engineering approaches provide unparalleled flexibility to control the composition, size,

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shape, surface area and surface chemistry, and functionality of nanostructures that can be
used to develop a new generation of modified materials or to coat existing solid surfaces for
biofilm prevention. Functionalized nanoparticles, including stimuli-triggered activation, can
be designed to enhance penetration and selectively target or release drugs locally after
bacterial attachment or within biofilms. In this Review we focus on overall concepts and
provide insights into their clinical potential based on recent studies using in vivo models. We
have provided a full list of current and prospective technologies and additional references in
Supplemental Table S1.
Surface modifications
Surface-tethering or the incorporation of an antibiotic or biocide within a “reservoir” coating
has long been studied as a possible approach to inhibit bacterial adhesion and biofilm
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formation110. However, sustaining efficacy and therefore justifying their progression to wide
scale use into the clinic has been challenging. The amount of biocide required to achieve
efficacy as well as its chemical composition are often limited by potential deleterious effects,
exemplified by silver nanoparticles which were shown to be toxic to rat hosts111.
Additionally, antimicrobial reservoir coatings are subject to progressive decreases in efficacy
as the active agent is depleted and thus have a limited lifetime of activity. Further,
nonspecific absorption of exogenous surfactants and proteins may mask the engineered
surface or hinder release.
Advances in material and surface engineering have led to the development of well-defined
topographic surface patterns that can control biofilm formation without including
antimicrobial agents112. The most well-established ordered topography is the Sharklet™
surface. Inspired by shark skin and its inherent anti-biofouling properties, microscale ribs of
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various lengths are combined into a repeating diamond micropattern, creating a textured
surface that prevents macro and micro biofouling113 as well as bacterial colonisation and
biofilm formation when incorporated into the surfaces of medical devices114. Surface
modifications are mostly focused on nonspecific protein repulsion and the inhibition of
bacterial colonization. This can be challenging due to the structural and physio-chemical
diversity of the numerous proteins in biological fluids surrounding a surface in a biomedical
setting 115. Hydrophilic polymer brushes or tethered polymers such as poly(ethylene glycol)
(PEG) are widely used in the prevention of medical device fouling116. While early bacterial
adhesion is attenuated, probably due to the inhibition of an initial protein priming layer, the
multifaceted nature of bacterial colonization (often involving non-proteinaceous adhesins)
can lead to eventual biofilm formation117. Further studies with ‘super-hydrophilic’ (super-
wet) or ‘super-hydrophobic’ surfaces have decreased protein deposition and bacterial
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attachment of clinically relevant surfaces115,118.
The incorporation of these materials into medical devices shows promising, but variable
results. While recently greater sustainability of super-hydrophobic surfaces upon mechanical
abrasion has been demonstrated119, the antibiofilm effects of these surfaces are often
transient or subject to species bias120. Short to medium-term biofilm suppression may be
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sufficient however to permit effective immune and prophylactic responses and tissue
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integration of a foreign body.
The development of functionalized medical implant surfaces with a vast array of

antimicrobial and antibiofilm properties has been intensely studied, particularly with respect
to titanium implants. To use these surfaces in biomedical applications, modern surface
design has been largely driven by top-down methods such as lithography, imprinting and
others121 to produce a vast array of antibacterial coatings, including but not limited to, silver,
copper, titanium dioxide and chitosan. Furthermore, emerging bottom-up approaches using
nanomaterials as ‘building blocks’122 and surface attachment and immobilization of
biomolecules, including antimicrobial peptides or proteins and polysaccharides123,124, have
also resulted in the development of antibacterial surface coatings. Moreover, the unexpected
discovery that certain bacterial polysaccharides can inhibit biofilm formation124, has led to
the development of strategies to counter biofilm formation‥ Hyaluronic acid (which is one
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of the most studied polysaccharides) reduced the adhesion of S. aureus to hyaluronic acid-
coated titanium surfaces125 and poly(methyl methacrylate) intraocular lenses126.
Bottom-up surface-assemblies can also be combined with top-down surface processing127 to

generate nanocoatings with biofilm-targeting properties and biocompatibility128. Recent in
vivo studies demonstrated the feasibility and efficacy of tunable multi-layer nanocoatings
that released different combinations of antibiotics129 or sequential delivery of gentamicin
and an osteoinductive growth factor130 in a time-staggered manner for prevention of biofilm-
associated infection and bone tissue repair around implants. Both studies demonstrated the
ability to prevent biofilm formation on the device surface, relative to uncoated controls, with
the nanocoatings able to clear infiltrating bacteria and prevented colonization of the implant
while promoting bone formation and osseointegration. Importantly, biocompatibility, as well
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as long-term host retention and release kinetics were also demonstrated, thus providing
promise for their more wide-spread application in orthopaedics.
Efforts to engineer surfaces with even more control over the specificity and sensitivity of
their antibacterial or antibiofilm capabilities have led to so-called smart surfaces, which are
also known as stimuli-responsive or triggered biofilm-targeting surfaces. Triggers, including
pH, temperature, salt concentration, metabolites, electrical currents and photoactivation,
induce topographical and chemical changes in the surface area as well as generating heat or
induce drug release to kill or repel bacterial attachment (Supplemental Table S1). The design
principles for controlling bacterial adhesion or biofilm removal mechanisms that may be
triggered on demand are intriguing. However, the effectiveness of these approaches has been
evaluated largely in vitro. Consequently, as with all surface modifications, whether
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functionality will remain in vivo upon binding of endogenous host proteins in saliva, blood,
synovial fluid and urine is unclear. Another consideration is that bacteria that associate with
the surface may be killed and remain attached, thus masking the underlying technology and
even provide a nutrient source for other bacteria. Therefore, it is important to not only have a
killing effect but also a ‘self-cleaning’ mechanism, possibly facilitated by mechanical shear
of surrounding body fluids or tissues. Furthermore, challenges to enhance mechanochemical
stability and overcome coating deterioration and dissolution, and non-adverse host reactions
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to the coating itself will need to be addressed in future studies to facilitate clinical
application131–133.
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Nanoparticles
Nanoparticles are versatile and bioactive and they are becoming increasingly popular as an
biofilm-targeting approach. Nanoparticles with intrinsic antimicrobial activity, primarily
inorganic materials such as silver, can act as biofilm-targeting agents or as nanocoatings (as
described above). Due to their flexible chemical structures, they can also function as drug
delivery vehicles (nanocarriers) with organic nanoparticles accounting for over two-thirds of
the systems approved for use in humans134. Furthermore, both inorganic and organic
nanoparticles can be combined or modified by adding molecules (hybrid nanoparticles) to
enhance their biological properties or provide multifunctionality. As excellent in-depth
reviews on the principles and current applications of nanoparticles, particularly silver, are
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available13,135, we focus on clinically used liposomal nanoparticles for drug delivery and
emerging technologies, including stimuli-triggered activation, that have shown efficacy in
vivo.
Liposomes are physiologically compatible vesicles that are composed of one or more
phospholipid bilayers, and they represent one of the most widely developed organic
nanoparticles for drug delivery . They are able to penetrate the biofilm well, are
biocompatible and show efficacy against biofilms of a wide variety of bacterial species for a
diverse number of antibiotics136,137. These nanocarriers can protect the antimicrobial agent
from deleterious interactions with the matrix, or enzymatic inactivation and degradation at
the infection site by other bacterial and host components. The lipid structure can also fuse
with the bacterial outer membrane releasing the drug directly into the cell, thereby
potentially maximizing therapeutic effects while reducing host cytotoxicity137. Furthermore,
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liposomes can carry more than one drug by co-encapsulation and can be also functionalized
by linking biomolecules (for example, peptides, pH-responsive polymers) on the
nanoparticle surface to increase targeting specificity and triggered release. Importantly,
however, some studies have reported a reduced efficacy of liposomal-encapsulated
antimicrobials dependent on the environment in which the biofilm resides; for example:
host- and microorganism-derived substances such as mucus and alginate could inhibit
bacteria-liposome interactions138. Nevertheless, several formulations are currently in
preclinical studies and clinical trials, and some are commercially available 139. For example,
liposomal ciprofloxacin and amikacin have shown promise in the management of chronic
lung infection in cystic fibrosis140,141. The potential of liposomes to function as delivery
agents for other antimicrobials, such as NO, has demonstrated significantly reduced S.
aureus biofilm mass compared to controls in a sheep model of chronic rhinosinusitis142.
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Whilst this study did not note any negative clinical symptoms, a transient increase in heart
rate and decrease in mean arterial pressure was observed in the animals which require
further investigation before this strategy can advance into human trials.
Nanoparticles with multi-functionality or on-demand activation upon specific stimuli similar

to smart surfaces represent the most widely developed class of nanoparticles currently under
development (Supplemental Table S1). Recent studies with inorganic nanoparticles such as
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iron oxide (Fe3O4) with a peroxidase-like function catalyzed hydrogen peroxide (H2O2) at
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concentrations ranging from 0.1–1% H2O2 in a dose- and pH-dependent manner, and
showed potent effects against virulent oral biofilms in vivo143. Under acidic (pathological)
conditions, nanoparticles activated the generation of free radicals from H2O2 in situ, which
induced the degradation of the biofilm matrix and rapid killing of the embedded bacteria
(>5-log reduction of viable cells compared to control cells within 5 min, and 5000-fold more
effective than 1% H2O2 alone)144. Daily topical treatments effectively reduced the onset and
severity of dental caries (tooth decay), preventing cavitation altogether in a rodent model of
the disease. The pH-dependent functionality prevents catalytic reaction at physiological pH
and unmitigated free-radical production, thus improving biocompatibility.
Stimuli-triggered mechanisms by nanoparticles can also enhance the selectivity of drug

activation or delivery to cells within a biofilm, protecting host tissues and the commensal
microbiota while targeting infective agents within pathological microniches143,145,146.
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Delivery of the antibacterial agent farnesol via acidic pH-triggered polymeric nanoparticles
enhanced its biofilm-targeting activity 4-fold (compared to farnesol alone); thus, the delivery
system greatly improved the drug efficacy against an oral biofilm infection in vivo following
topical treatment146. These water-soluble polymeric nanocarriers can encapsulate
hydrophobic and apolar drugs into aqueous solution, which is a crucial issue in product
development. Similarly, nanoparticles that are conjugated with a pH-responsive element145
or pH-sensitive surface charge switching147 were developed to increase biofilm penetration
and selective bacterial binding for targeted delivery and antibacterial activity in acidic
conditions.
Another exciting area of development is in increasing the specificity of the nanoparticles by

selectively targeting biofilm matrix constituents or through the introduction of bacteria-
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specific ligands, to improve both efficacy and biocompatibility. Nanoparticles functionalized

with biofilm EPS matrix-digesting enzymes (DNase) and loaded with ciprofloxacin
eradicated established P. aeruginosa biofilms without cytotoxicity against macrophages49.
Likewise, tobramycin alginate-chitosan nanoparticles functionalised by conjugation to
dornase alfa (DNase) demonstrated better biofilm penetration and DNA degradation in
sputum from patient with cystic fibrosis and increased protection against bacteria in an
invertebrate infection model148. Furthermore, nanoparticles that were designed to release
and activate NO in situ had antifungal and antibacterial effects, inhibited biofilm formation
and promoted the degradation of the EPS matrix in vitro and in vivo149,150. Recently, reports
showed that linking antimicrobial peptides102 or aptamers151 to nanoparticle surfaces
enhanced their killing efficacy, specificity or functionality.
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Overall, nanoparticles offer a promising therapeutic platform for the development of new
effective biofilm-targeting approaches. However, whilst the development of novel
nanoparticles has continued apace, there is a continually widening gap between the number
of new formulations under laboratorial investigation and those in clinical use. Further
advances in this field should focus on enhancing in vivo efficacy (compared to current
treatment modalities) and biocompatibility, and on understanding the potential toxicity and
the metabolism of nanoparticles in the body. Affordable large scale manufacturing would be
also required for product development to the healthcare market. Nevertheless, the
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availability of previously FDA-approved nanoparticles demonstrates their potential for more

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wide-spread future clinical use.
Future directions
The initiation of a biofilm involves complex and dynamic interactions among the surface,
the microorganism and the EPS. Upon biofilm establishment, the adhesive strength and
viscoelastic properties make the removal of a biofilm from surfaces difficult, and resident
microorganisms become tolerant to antimicrobials. Although tolerance is a common feature
of biofilms, the mechanisms underlying tolerance as a microbial survival strategy are
multifaceted. Likewise a reciprocal multifaceted approach to control biofilms is far more
likely to achieve clinical success than a futile search for a magic bullet (Box 2).
Understanding the complexity of biofilm biology highlights the role of complementary
strategies that target both the microorganisms and the surrounding EPS matrix to either
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prevent the initiation of a biofilm or to disrupt existing biofilms. The challenge of using
antimicrobials alone, which may kill microorganisms, but leave behind biodegradable
substrates for microbial reutilization, must be addressed. Thus, eliminating existing biofilms
may require simultaneous degradation of the protective EPS matrix, and targeting and killing
both resident microorganisms and dispersed cells. The complexity of polymicrobial
interactions (synergistic, cooperative or antagonistic), spatial organization and community
behaviour with host immunity factors further reinforces the need of a combinatorial therapy.
Rapid advances in drug discovery methods should accelerate the identification of EPS-
inhibitors, inducers of biofilm dispersal and agents that target dormant cells, as well as
combinations thereof with host immunomodulation therapies152,153. However, further
validation of proof-of-concept studies using clinically relevant animal models as well as
clinical trials are needed for rigorous evaluation. Bacterial co-cultures with primary human
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cells to evaluate host-microbial response can be valuable to investigate, for example, the role
of genetic mutations such as found in patients with cystic fibrosis or PCD on the
establishment and treatment of biofilms in patients suffering from these diseases.
Opportunities to create physico-chemical and biological structures, including organ-on-chip,
within microfluidic devices or using 3D printed tissues may also help assess treatment
efficacy by mimicking in vivo-like environments.
Furthermore, new technologies , including ‘smart-release’ or ‘on-demand activation’ of

bioactive agents when triggered by pathogenic microenvironments (e.g. acidic pH or
hypoxia), have been developed for enhanced selectivity and controlled in situ drug delivery.
However, the vast majority of the studies were conducted in vitro using non-clinically
relevant models or treatment regimens, with many failing to progress to in vivo studies and
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even fewer to clinical application. The complexity of the host microbiota, where
commensals co-exist with potential pathogens, provide a great challenge in developing
antimicrobial agents against a particular microbial species. The presence of biological fluids
that change surface chemistries poses yet another challenge. The ability of a drug to
penetrate existing biofilms should be also considered, as this feature affects both potential
cytotoxicity and antibacterial efficacy, and the potential for de novo emergence of
antimicrobial resistance (owing to bacteria being subjected to sub-lethal antibiotic
concentrations). Antibodies, aptamers or peptides that are linked to nanoparticles greatly
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enhance specificity, although higher costs and additional chemistry to produce multi-
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component structures may be limiting factors. A key approach may be to trigger

antimicrobial activity in response to pathogenic microenvironments (for example, acidic pH,
hypoxia or pathogen-derived metabolites). Thus, the biological effects can be tuned to
specifically target the biofilm microenvironment, degrade the matrix and kill resident
bacteria, thereby eradicating the pathogenic niche with precision and minimal cytotoxicity to
surrounding tissues. Nevertheless, we noted a discrepancy between the research efforts on
new technologies and commercialization. A concerted effort of chemists, engineers and
biomedical researchers combined with toxicology and safety studies will help clinicians to
assess the efficacy of these new technologies in clinical trials. However, the successful
translation into the clinic is not just dependent on efficacy of the technology, but also on
regulatory agencies and industry efforts to bring it to the market. Future directions should
focus on achieving maximal efficacy and specificity with minimal toxicity and long-term
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therapeutic effects along with industry partnerships to develop low-cost and practical
formulations for clinical use.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Work in the authors laboratory is supported in part by the National Institute for Dental and Craniofacial Research
grants DE018023, DE025220 and DE025848 (HK); The Ohio State University Infectious Disease Discovery
Theme- Public Health Preparedness for Infectious Disease Transdisciplinary Team Grant (PS).
P.S. has received research funding from and/or has consulted from Philips Oral Healthcare, Smith & Nephew,
Biocomposites Ltd., Zimmer-Biomet, Colgate-Palmolive. H.K. has received funding from Johnson&Johnson,
Author Manuscript
Colgate-Palmolive and DENTSPLY. RPH has consulted for Biocomposites Ltd.
Glossary
Lock therapy
An approach whereby a high concentration of antibiotics are injected into the catheter lumen
for an extended period to eradicate bacteria. Catheter locks have been used to treat sepsis
since the 1980s; however with the understanding that infecting microorganisms are present
as biofilms on medical device materials, this approach is now specifically tailored to
improve efficacy
EPS
The EPS can contain exopolysaccharides, fibrous and globular proteins (including
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extracellular enzymes), lipids and nucleic acids (eDNA). Those components form a matrix
that can be surface-associated or secreted locally or deposited on abiotic and biotic surfaces.
The EPS-matrix acts as a ‘multifunctional scaffold’ that supports and protects embedded
bacteria
Nitric oxide
Nitric oxide (NO) is a ubiquitous signalling molecule found in both prokaryotic and
eukaryotic systems. NO is toxic in the mM range, but in the pM and nM range it can be used
Koo et al. Page 19
to form oxidative and nitrosative reactive species that interact with proteins, DNA and
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metabolic enzymes. As NO is labile, the optimal concentration to disperse biofilms is

difficult to measure; however NO microelectrodes are highly sensitive and offer excellent
spatial and temporal resolution in tissues or body fluids
Antimicrobial peptides
A subset of host defence peptides with antibiotic activity. Peptides such as LL-37
(cathelicidin) and human β-defensins are rapidly-acting, small-molecule effectors as part of
the innate immune response of the host
Topographic surface patterns

Patterns include protruding squares, cone-shapes, wrinkle and ridge-like patterning or
nanopores that disrupt bacterial adhesion
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Super-hydrophobic surfaces
Surfaces that maintain air at the solid-liquid interface when hydrated. This leads to improved
functionality via water repellency or reduced drag
Smart surfaces
Smart surfaces elicit their effect only upon contact with certain physiological or
physiochemical cues to provide targeted application, thus increasing therapeutic precision
and reducing the risk of cytotoxicity
Nanoparticles
Structures with a size range between 1–1000 nm. They can be classified as organic or
inorganic and can exhibit antibacterial properties or can be used as drug delivery systems
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Adhesins
Bacterial or fungal surface-associated determinants that mediate adherence to living cells or
attachment to abiotic surfaces and can promote virulence
Antimicrobial chemotherapy
The clinical treatment of microbial infections with antimicrobial agents
Mannosides
A mannose glycoside consisting of a carbohydrate bound to the hydroxyl group of another
compound by O-, N-, S- or C-glycosidic bonds, each with different susceptibilities to
hydrolysis
Curli
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A class of bacterial amyloid (aggregates of proteins that form insoluble fibres) produced by
many Enterobacteriaceae and a major component of the extracellular matrix, promoting
surface adhesion, cell aggregation, and biofilm formation
Type-I pili
Filamentous surface structures possessing a FimH adhesin at the pilus tip, mediating
adherence to host cells and uropathogenic E. coli invasion of bladder epithelial cells
Koo et al. Page 20
Cystic fibrosis transmembrane conductance regulator

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(CFTR). A transmembrane protein and ion transport channel that regulates epithelial fluid
homeostasis central to airway mucociliary clearance and defence against inhaled pathogens
Biguanides
Class of organic compounds (C2H7N5) used as oral antihyperglycemic drugs. Derivatives of
this compound with bactericidal activity are commonly used as antiseptic and disinfecting
agents such as chlorhexidine
Surfactants
Compounds that lower the surface tension between liquids and solids. Surfactants are used
as cleaning detergents and some biofilm bacteria produce their own surfactants in order to
disperse from a surface
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Biofouling
The unwanted accumulation of micro and macro-organisms on surfaces. Microbial biofilms
are often considered ‘biofouling’, particularly in the context of industrial surfaces;
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Text Box 1. Common features of microbial biofilms

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Chemical composition, physical properties

Adherence. Microorganisms adhere to virtually all man-made materials (i.e. plastics, metals, ceramics and
hybrids) and biotic surfaces (i.e. tooth enamel, bone, skin, airway, intestinal, and vaginal mucosa, connective
tissue, vascular endothelium), using both specific (bacterial adhesin-host receptor interactions) and non-specific
adhesion (hydrophobic or electrostatic forces) mechanisms154.
Extracellular polymeric substance (EPS) matrix. Although the precise chemical and physical composition of
the EPS (polysaccharides, proteins, nucleic acids) varies between species and growth conditions (i.e. nutrient
type and abundance, hydrodynamics, temperature, oxygen concentration), the EPS provides a scaffold for
mechanical stability, and creates compartmentalized chemical and physical microenvironments affording
protection to the cells within a heterogeneous 3D structure.
Architecture. Although there is some variation in the structure of in vitro grown biofilms, there are a limited
number of common forms (flat patches, mounds, mushrooms, towers, ripples, streamers) that are not generally
species specific but largely dependent on biofilm maturity and the production of certain EPS components and
growth conditions. Biofilms seen in many clinical specimens tend to consist of aggregates of cells of varying
sizes and mixed-species in polymicrobial systems 155.
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Viscoelasticity. A material property that allows biofilms to absorb and dissipate energy, rather than detach,
when exposed to mechanical forces, such as hydrodynamic shear. The elastic component allows the biofilm to
spring back into shape during intermittent perturbations, while the viscous component allows biofilms to flow
like liquids when forces are sustained.5.
Heterogeneity. Biofilms are heterogeneous (non-uniform) in distribution, structure and physiology at various
spatial scales. On the larger scale (mms to cms) they are generally not uniformly distributed on surfaces but
occur in patches of cell-clusters (also called microcolonies or aggregates) in a range of sizes and shapes. Within
the biofilm heterogeneous and compartmentalized microenvironments (10s to 100 µms) develop which
modulate microbial activity, intercellular signalling and metabolic exchange locally, thus spatially organizing
cellular and communal behaviour which is an important factor for enhanced tolerance and persistence.
Physiological and regulatory aspects
Developmental life cycle. Pseudomonas aeruginosa as a widely used model organism for studying microbial
biofilms formation. Although many of the concepts identified in this organism, such as the mechanism of
attachment, growth, maturation and dispersal, are widely conserved among other biofilm forming pathogens,
there are fundamental differences. For example, many biofilm-forming species, such as staphylococci, unlike P.
aeruginosa, are non-motile, many species have surface structures that are important for adhesion (capsule, pili
or flagella) but many do not, and lastly not all species have known signalling systems.
Diffusible cell signals. These signals co-ordinate population behaviour156, metabolic activity, biofilm
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formation and dispersal. Families of homoserine lactones are produced by a number of Gram negative species
while Gram positive organisms more commonly use autoinducing peptides (AIP).
Altered microenvironment formation. The development of gradients in nutrients, pH and oxygen as a

consequence of metabolic activity of microorganisms that reside in a biofilm and diffusion limited mass
transport of molecules into and out of the EPS matrix alter the microenvironment in the biofilm.
Dormant or slow growing sub-populations. Those include persisters and small colony variants (SCVs),
which are tolerant to antibiotics. In addition, nutrient depletion in the interior of the biofilm can result in a
stationary phase-like dormancy.
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Text Box 2. Lessons learned from cancer

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Over 100 years ago Paul Ehrlich, the German physician scientist used the term ‘magische
kugel’ to describe an ideal hypothetical therapeutic agent that specifically targets and
kills disease-causing cells157. In the context of cancer’ the target of such a magic bullet
were the newly discovered receptors found on tumor cells. Another magic bullet was
interferon, the cytokine that was discovered in 1957158. However, a magic bullet for
cancer therapy remains elusive in part because over the last 40 years of intense molecular
research, it has been shown that “cancer is not simply a single disease that affects many
parts of the body”. Rather, it is many different diseases with common themes that can
cause different kinds of disorders in many of our organs”159. In addition, individual
tumors exhibit substantial chemical and clonal heterogeneity with distinct phenotypes
which combined with the ability of cancer cells to rapidly adapt to chemotherapeutics
and the microenvironment, challenge both broad spectrum and targeted therapies. In
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tumors, the structure and composition of the extracellular matrix is often altered, creating
a favourable cellular niche for malignant transformation and cancer progression. Biofilms
share similar common themes (Box 1), including the ability to create distinct
microenvironments with unique chemical, physical, phylogenetic, genotypic and
phenotypic heterogeneities. Early cancer therapy borrowed from approaches to treat acute
bacterial and viral infections by targeting individual cells (with antibiotics and
vaccination), with limited success. Our current understanding of biofilm biology is
following a similar path to tumor biology. Rather than piled-up assemblages of clonal
cells, microbial biofilms represents a dynamic self-constructed ecosystems within a
matrix containing highly heterogeneous and compartmentalized milieu, and more
effective biofilm therapies will likely need to target the complete microenvironment as
well as the individual cells within144.
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Text Box 3. Persistence, resistance and tolerance

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The terms persistence, resistance and tolerance, are often used interchangeably when
used to describe the inability of antibiotics (and antimicrobial agents) to inhibit or kill
bacteria within a biofilm to the same extent as planktonic cultures 160. Resistance usually
has an underlying heritable genetic basis that might be acquired through point mutation
or horizontal gene transfer and is defined through standardized MIC and MBC assays.
Tolerance is less-well defined and is arguably more appropriately used when antibiotic-
susceptible strains (by MIC and MBC) require much higher concentrations to obtain
similar log-reductions when growing in the biofilm phenotype. Importantly, tolerance can
be lost when biofilms are dispersed into single cells, thus dispersal strategies are
normally considered as adjuvants for antimicrobial therapy. However, dispersed
planktonic aggregates of cells may still retain tolerance. Persistence (and persistent
biofilm) is a term that is loosely used to describe a clinically protracted unabated biofilm
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infection despite treatment. However, persistence in this sense should not be confused
with ‘persisters’ 161 or sub-populations of cells with a distinct dormant phenotype
affording them protection against antibiotics, which kill the metabolically active
population. Persister cells can occur in both planktonic and biofilm cultures, but the
stressful conditions, physical stability and protection from host phagocytes afforded by
the biofilm microenvironment appear to contribute to harbouring microbial populations,
which grow and repopulate once the antibiotic stress is removed. It is thought that these
populations are tolerant of conventional antibiotics because there are no active cellular
processes to interrupt. These subpopulations can form spontaneously or be induced from
environmental stresses in the biofilm microenvironment162 (see Ref.161 for a Review).
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Figure 1. Opportunities for therapeutic intervention during various stages of the biofilm life-
cycle
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Biofilm formation proceeds as a developmental process with distinct stages: “initial

adhesion” where microorganisms bind to host or medical device surfaces through cell
surface associated adhesins; “early biofilm formation” where they begin to divide and
produce EPS which enhances adhesion, while forming the matrix that embeds the cells;
“biofilm maturation” where 3D structures develop in which the EPS matrix provides a multi-
functional and protective scaffold which allows heterogeneous chemical and physical
microenvironments to form where microorganisms co-exist within polymicrobial and social
interactions (competitive and synergistic); and finally “dispersal” where cells leave the
Koo et al. Page 33
biofilm to re-enter the planktonic phase. Biofilms can be targeted at these various stages. a)
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The initial phase of biofilm formation can be disrupted, for example, by preventing the
attachment of microorganisms by interrupting the interactions between the microorganism
and the surface, by targeting cell surface associated adhesins (appendages, proteins and
EPS). b) The inhibition of early stages of biofilm development includes targeting the
production of EPS and cellular division. c) Disruption of formed biofilms could be achieved
by physical removal, the degradation of the EPS-matrix, targeting the establishment of
pathogenic microenvironments (low pH or hypoxia) and social interactions (in
polymicrobial biofilms) as well as elimination of dormant cells. d) Finally, biofilm
dispersion can be induced by EPS matrix remodelling or activation to dispersal mechanisms.
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Koo et al. Page 34
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Figure 2. Targeting the EPS

Disruption of EPS components, and the underlying mechanisms that are responsible for the
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production and secretion of EPS components, represent attractive targets for the
development of biofilm-targeting strategies, some of which have potential efficacy across
microbial species. One approach includes the degradation of the EPS. Treatments have been
developed that directly target the eDNA (DNases), exopolysaccharides (dispersin B,
glycoside hydrolases, monoclonal antibody vaccines), and protein (DNABII family
antibodies) components of the matrix. EPS adhesin-binding antibodies or inhibitors and
phage-encoded peptidoglycan hydrolases have been developed to target bacterial adhesion
Koo et al. Page 35
and biofilm initiation. Inhibitors of EPS synthesis and the secretion systems have also shown
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promise to disrupt biofilm accumulation. Endogenous pathways that induce biofilm dispersal
can also be targeted, including the regulation of c-di-GMP and c-di-AMP levels using
exogenous NO and inhibitors, or targeting quorum sensing using various inducing peptides
and messenger molecules. Importantly, all of these treatment strategies, alone or in
combination, can lead to inhibition of biofilm formation, disrupt biofilm integrity and/or
promote the release of individual bacterial cells that are more susceptible to conventional
antibiotic treatment enhancing clinical efficacy.
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Koo et al. Page 36
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Figure 3. Technological approaches to combat biofilms

Recent advances in material science and nanotechnology enabled the engineering of a wide
array of biofilm-targeting strategies. a) The material and surface properties of medical
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devices, such as surface charge, hydrophobicity, roughness, topography and chemistry

among others, can be modified to prevent bacterial attachment and therefore attenuate or
block biofilm formation. Additionally, ‘smart’ or stimuli-triggered responsive surfaces can
be constructed that elicit their effect only in response to physical contact with cell-wall or
membrane associated adhesins or chemical cues (i.e. secreted EPS, metabolites) of the
bacteria. b) Advancement in nanoparticle synthesis has led to the development of diverse
approaches to combat biofilms. Inorganic metallic (silver, copper etc.) and organic
nanoparticles (liposomes, aptamers etc.), have been increasingly evaluated to improve their
anti-biofilm efficacy, as well as their biocompatibility to reduce toxic effects on the host.
Nanoparticles can be used to form nanocoatings, be incorporated into materials as
composites or fillings or combined together with conventional antimicrobials and other
approaches designed to physically disrupt or remove the biofilm. Furthermore, antimicrobial
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peptides (AMPs) and aptamers also display specific biofilm-targeting properties that can be
also used to enhance specificity and efficacy of nanoparticles (hybrid nanoparticles). c) New
technologies for physical biofilm removal, including mechanical, energy- and light-based
disruption, may further improve biofilm intervention strategies. Given the multifaceted
nature of biofilm formation and the complex microbial interactions with the surrounding
physical and chemical environment, a combination of these approaches may be required to
successfully combat biofilm-mediated disease.
Koo et al. Page 37
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Figure 4. Multi-targeting approach to combat biofilms

The physical and biological complexity of biofilms and tolerance to antimicrobials render
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them less susceptible to conventional therapeutic approaches. Biofilm targets include

microbial cells (often polymicrobial communities) and the EPS matrix, and therapeutics can
be delivered from the overlying surrounding biological fluid as well as the surfaces of the
medical devices themselves. We envision exogenous approaches (such as adhesion-targeting
materials and coatings, and adhesin-blocking agents) to complement or synergize with
endogenous activation (such as immunity modulation) to prevent microbial attachment to
host or abiotic surfaces in patients. Likewise, a combination of approaches that degrade the
protective matrix, activate dispersal, and target the resident pathogens, persisters and
dispersed cells without affecting commensals may be required to eliminate existing biofilms.
Long-term effects of modified surfaces in the presence of biological fluids as well as
enhanced drug penetration properties and a decrease in toxicity or allergic reactions are
required for in vivo efficacy. These combined with clinically relevant treatment regimen
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(either topical or systemic) and long-term effect assessment should help successfully
translate the hypothetical concepts into the clinic. The grey arrows indicate that biofilm
bacteria and EPS can move or interact between the surface and fluid phases.

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Targeting microbial biofilms: current and prospective

Pennsylvania, PA, USA.

Initially reported as an arcane behaviour of bacterial populations, microbial biofilm

role of the extracellular matrix in conferring antimicrobial tolerance to biofilms is being

Current therapeutic approaches

delivery compared to systemic administration. Several antimicrobial metal or inorganic

Disrupting EPS synthesis and secretion, and binding of EPS adhesins

These nucleotide-signalling molecules regulate glucan-producing exoenzymes (for example,

The inhibition of EPS glucan synthesis by glucosyltransferase using small-molecule

Targeting EPS chemical composition and structure

multiple bacteriophage-encoded endolysins, each with unique actions, all added

hydrolases, glycoside-hydrolases and DNases enhanced antimicrobial delivery and

EPS-targeted antibodies and nucleic acid-binding proteins

Monoclonal antibodies against P. aeruginosa-derived EPS identified epitopes that bound to

Nonetheless, targeting broadly conserved components in EPS is desirable. The DNABII

Inducing biofilm dispersal

Targeting cyclic-di-GMP pathway

A well-characterized approach to modulate c-di-GMP levels is though nitric oxide (NO).

NO-donor instability is also an issue that is being addressed by developing nitroxides

Targeting quorum sensing

Another approach is based on evidence that iron metabolism is important in biofilm

Targeting dormant cells in biofilms

Antimicrobial peptides (AMP) represent another approach in treating biofilms independent

The promise of new technologies

engineering approaches provide unparalleled flexibility to control the composition, size,

attachment of clinically relevant surfaces115,118.

integration of a foreign body.

The development of functionalized medical implant surfaces with a vast array of

Bottom-up surface-assemblies can also be combined with top-down surface processing127 to

Nanoparticles with multi-functionality or on-demand activation upon specific stimuli similar

Stimuli-triggered mechanisms by nanoparticles can also enhance the selectivity of drug

Another exciting area of development is in increasing the specificity of the nanoparticles by

specific ligands, to improve both efficacy and biocompatibility. Nanoparticles functionalized

availability of previously FDA-approved nanoparticles demonstrates their potential for more

wide-spread future clinical use.

Furthermore, new technologies , including ‘smart-release’ or ‘on-demand activation’ of

component structures may be limiting factors. A key approach may be to trigger

Colgate-Palmolive and DENTSPLY. RPH has consulted for Biocomposites Ltd.

metabolic enzymes. As NO is labile, the optimal concentration to disperse biofilms is

Topographic surface patterns

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