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Current Topics in Medicinal Chemistry, 2017, 17, 1-8 1
REVIEW ARTICLE
1568-0266/17 $58.00+.00 © 2017 Bentham Science Publishers
Eradicating Bacterial Biofilms with Natural Products and their Inspired
Analogues that Operate Through Unique Mechanisms
Aaron T. Garrison and Robert W. Huigens III*
Department of Medicinal Chemistry, Prof. Robert W. Huigens III, University of Florida, Gainesville, USA
A R T I C L E H I S T O R Y
Received: March 20, 2014
Revised: April 16, 2014
Accepted: April 20, 2014
DOI: 10.2174/156802661766616121
4150959
Abstract: Bacterial biofilms are surface-attached communities of slow- or non-replicating bacterial
cells that display high levels of tolerance toward conventional antibiotic therapies. It is important to
know that our entire arsenal of conventional antibiotics originated from screens used to identify inhibi-
tors of bacterial growth, so it should be little su rprise that our arsenal of growth-inhibiting agents h ave
little effect on persistent biofilms. Despite this current state, a diverse collection of natural products
and their related or inspired synthetic analogues are emerging that have the ability to kill persistent
bacterial biofilms and persister cells in stationary cultures. Unlike conventional antibiotics that hit bac-
terial targets critical for rapidly-dividing bacteria (i.e., cell wall machinery, bacterial ribosomes),
biofilm-eradicating agents operate through unique growth-independent mechanisms. These naturally
occurring agents continue to inspire discovery efforts aimed at effectively treating chronic and recur-
ring bacterial infections due to persistent bacterial biofilm s.
Keywords: Bacterial biofilms, Persister cells, Natural products, Biofilm-eradicating agents, Drug discovery.
1. INTRODUCTION
Robert Koch won the Nobel Prize in 1905 for his work
on tuberculosis (TB) [1,2]. Koch also contributed to the
germ theory of disease stating that some diseases are caused
by microorganisms, too small to see without magnification,
that invade human hosts [3]. Two decades later, Alexander
Fleming discovered that a fungus had contaminated and
killed a culture of Staphylococcus aureus that was growing
on a petri dish [4]. The 1945 Nobel Prize was awarded for
the discovery of penicillin, an effective human therapeutic
and microbial warfare agent that a fungus uses to kill bacte-
ria. Over the last 75 years, we have continued to harness
Nature’s antibiotics (microbial warfare agents) to treat life-
threatening bacterial infections in humans [5,6]. We have
relied heavily on naturally-occurring antibiotics (e.g., peni-
cillin) and synthetic medicinal chemistry to optimize genera-
tions of analogues as enhanced antibiotic agents (e.g., methi-
cillin) to improve our ability to fight bacteria [7]. Our current
arsenal of “antibiotics” consists mainly of naturally occur-
ring agents with a few antibacterial agents of synthetic ori-
gins (i.e., fluoroquinolones) [6].
It is important to note that all clinically used antibi-
otic/antibacterial agents were initially discovered as growth
inhibitors against rapidly-dividing planktonic (or free-
floating) bacteria [6]. Although this approach has been suc-
*Address correspondence to this author at the Department of Medicinal
Chemistry and Center for Natural Products, Drug Discovery and Develop-
ment (CNPD3), Assistant Professor at the University of Florida,
Gainesville, FL 32610, P.O. Box: 100485, USA;
Tel/Fax: 352-273-7718, 352-392-9455; E-mail: rhuigens@cop.ufl.edu
cessful in the discovery of clinically useful growth-inhibiting
agents, little progress has been made in identifying agents
that can kill surface-attached biofilms, which exist in a slow-
or non-replicating state [8]. During infection, planktonic
cells use a signaling process known as quorum sensing to
coordinate the simultaneous attachment to a surface (i.e.,
living tissue) [9-11]. Following initial attachment, bacterial
colonies develop and mature into densely packed biofilms
that are composed of viable yet metabolically dormant bacte-
ria housed within an extracellular matrix of biomolecules
[10,11]. Specialized non-replicating persister cells live
within bacterial biofilms, demonstrate high levels of antibi-
otic tolerance and are the underlying cause of chronic and
recurring infections (Fig. 1) [12-16].
Over the last several decades, our scientific community
has gained considerable insights regarding the basic biology
and medical relevance of bacterial biofilms [9-11,17,18];
however, most pharmaceutical companies have withdrawn or
significantly downsized their antibacterial discovery pro-
grams due to an unproductive antibiotic pipeline [19].
Biofilms occur in ~80% of all bacterial infections [20] and
are credited with >500,000 deaths each year in the United
States alone [21]. Persistent, surface-attached bacterial
communities play a critical role in numerous types of infec-
tions and diseases, including: hospital-acquired infections
(e.g., Staph infections) [22], osteomyelitis [23,24], endo-
carditis [23,24], p eriodontitis [24], skin/burn wounds [25-
27], implanted medical devices (prosthetic joint [28,29],
heart valves [30]), catheter-based infections [31,32] (i.e.,
urinary catheters [24], central venous catheters [33], cerebro-
spinal fluid shunts [34]), immunocompromised patients [35],
2 Current Topics in Medici nal Chemistry, 2017, Vol. 17, No. 14 Garrison and Huigens III
caries (tooth decay) [36] and lung infections in Cystic Fibro-
sis patients [23,24].
Innovative strategies must be implemented to identify
compounds that eradicate biofilm cells through growth-
independent mechanisms. Considering our own history of
antibiotic discovery, it stands to reason that microbial war-
fare agents include biofilm-eradicating agents which we
have not yet harnessed for therapeutic purposes. This review
covers natural products or natural product-inspired small
molecules that operate through mechanisms that kill biofilm
cells or persister cells in stationary planktonic cultures from
2009 to 2016. Excellent reviews have recently described
biofilm-inhibiting and biofilm-dispersing agents (anti-
biofilm agents that are not biofilm killers) [37,38] and will
not be presented in this review.
2. CALIBRATING PERCEPTIONS OF BIOFILM AND
PERSISTER CELL ERADICATION ACTIVITY IN
VITRO
When new antibacterial agents are presented, the first
data used to calibrate the effectiveness of the new agent is
the minimum inhibitory concentration or MIC value. The
MIC value corresponds to the lowest concentration of an
antibacterial agent required to completely inhibit the plank-
tonic (exponential) growth in vitro following overnight incu-
bation (Fig. 2). Compounds are typically tested in 2-fold
serial dilutions in 96-well plates, so a range of values are
common; however, it is accepted that a new compound with
an MIC of 1 µg/mL is indeed a potent antibacterial agent, or
planktonic growth inhibitor.
Fig. (2). Bacterial growth curve indicating various phases. Station-
ary phase cultures have higher populations of persister cells.
Although an MIC value is ideal for gauging planktonic
growth inhibition, how do we calibrate our perceptions of
biofilm or persister cell eradication? Since few compounds
have been reported with biofilm-killing activity, this can be
challenging. Biofilm eradication susceptibility assays have
Fig. (1). a) Historical timeline that includes the germ theory, antibiotic discovery and bacterial biofilms. b) Illustration of biofilm formation
from planktonic bacteria and subsequent biofilm maturation.
Eradicating Bacterial Biofilms with Natural Products Current Topics in Medici nal Chemistry, 2017, Vol. 1 7, No. 14 3
been developed, but are not as trivial as more standardized
growth inhibition (MIC) experiments. The in vitro effective-
ness of a biofilm-eradicating agent is determined by its
minimum biofilm eradication concentration (MBEC) value.
Biofilm eradication assays are conducted in Calgary
Biofilm Devices (CBD) [39] or standard 96-well plates [40].
Unlike MIC assays which determine a compound’s ability to
inhibit bacterial growth, biofilm eradication (MBEC) assays
have multiple phases, including: 1.) biofilm establishment
phase (biofilms are established on surfaces without test com-
pound), 2.) biofilm challenge phase (test compound is added
to established biofilms; active biofilm-eradicating agents kill
biofilms during this phase) and 3.) biofilm recovery phase
(test compound is washed away, biofilm growth and “recov-
ery” occurs in fresh media which results in turbid wells due
to viable biofilm dispersion and subsequent bacterial growth)
[8,39,40]. Bacterial biofilms that are eradicated during the
biofilm challenge phase 2, will result in non-turbid (no bac-
terial growth) wells after phase 3 as eradicated biofilms are
unable to grow and disperse cells into the fresh media to give
a positive turbidity result.
Calgary Biofilm Devices allow bacterial biofilms to es-
tablish on pegs that are attached to the inside lid of a 96-well
plate [39]. These pegs can easily be washed and transferred
to either the challenge or recovery plates during biofilm
eradication assays. Using the CBD, one can obtain relative
planktonic and biofilm eradication activities as minimum
bactericidal concentrations (MBC; planktonic killing) and
minimum biofilm eradication concentrations (biofilm kill-
ing) in a single assay [8,39]. This is a major advantage of
using CBD to assess a biofilm-eradicating agent and we have
demonstrated that biofilm-eradicating agents report
MBC:MBEC ratios of 1-3, while the rare conventional anti-
biotic that does show positive biofilm eradication activity
has MBC:MBEC ratios of >20 [8]. For instance, rifampicin
and doxycycline both reported MBEC values of 46.9 µM
despite demonstrating potent planktonic activities (MBC =
2.0 µM) against a MRSA clinical isolate using the CBD [8].
Against MRSA isolates (in the same study), vancomycin was
unable to eradicate (MRSA) biofilms (MBEC >2,000 µM)
despite potent planktonic killing (MBC = 3.0-7.8 µM) in the
same assay using the Calgary Biofilm Device, which is a
clear demonstration of the antibiotic tolerance of biofilms
[8]. Although few compounds have been reported to eradi-
cate bacterial b iofilms, a new biofilm-eradicating agent with
an MBEC ≤ 50 µM is worth pursuing [8,40-43].
Persister cell eradication has also been demonstrated in
kill kinetic experiments of stationary cultures [42,43]. Unlike
exponential phase cultures of bacteria, stationary cultures
have high populations of non-replicating persister cells
[44,45]. Effective persister cell killers typically eradicate ≥3-
log viable stationary cells at 4-10X MIC.
This review presents a diverse panel of natural products
that operate through unique mech anisms leading to effective
biofilm eradication (with reported MBEC values) or killing
of stationary cultures. Several compounds (e.g., halogenated
phenazines [8]) have been evaluated in biofilm eradication
assays and against stationary cultures.
2.1. Antimicrobial Peptide Inspired Compounds
Antimicrobial peptides (AMPs) have evolved as host-
defense strategies to protect from microbial infection. These
peptides, ranging in size from 2 or 3 to more than 59 amino
acid residues, have exhibited antimicrobial activity through
several mechanisms, many of which are not completely un-
derstood [46]. One caveat of the development of AMPs as a
viable therapeutic option is the prevalence of known AMPs
that demonstrate activity through transmembrane pore for-
mation and non-selective membrane lysis (Fig. 3a). How-
ever, selective membrane-active AMPs can distinguish be-
tween eukaryotic and bacterial cells on the basis of unique
membrane components (e.g., cholesterol, neutral outer mem-
brane charges, etc.) [47,48]. The targeting of cellular mem-
branes also offers the advantage of circumventing the devel-
opment of traditional bacterial resistance mechanisms. [49].
Furthermore, several known mechanisms of AMPs which are
independent of cell lysis include, but are not limited to: floc-
culation of intracellular contents, binding to nucleic acids,
and inhibition of cell wall, protein and nucleic acid synthesis
[50-53].
Multiple research groups have drawn inspiration from an-
timicrobial peptides to develop biofilm-eradicating agents.
Wuest and Minbiole have developed AMP-inspired Quater-
nary Ammonium Cations (QACs) that exhibit potent anti-
bacterial and biofilm eradication properties [40,54]. Their
analogue design strategy utilizes the incorporation of many
structural features found among AMPs using short synthetic
pathways, including specific combinations of cationic, am-
phipathic, and hydrophobic motifs. In particular, promising
biofilm eradication has been observed for PQ-8 and QAC-
10 (Fig. 3b) with MBEC values of 50 µM for both com-
pounds against S. aureus. Potent biofilm eradication activity
was also observed against E. faecalis with reported MBECs
of 50 µM and 25 µM for PQ-8 and QAC-10 respectively.
Although the cationic moieties of QACs are critical for
activity, investigation into the structure-activity relationships
of these compounds revealed that cationic character alone
does not bestow efficacy onto QACs, which can be seen
from the inactivity of compounds QAC-3 and QAC-4
(MBEC >200 µM against S. aureus and E. faecalis). Follow-
ing manipulation of the aliphatic linker length through ana-
logue synthesis, it was concluded that the spatial distribution
of cationic charges was inconsequential. QAC-10 demon-
strates significant haemolysis activity against red blood cells
[40], which is indicative of non-membrane selective lysis.
These studies have clearly demonstrated the potential for
eradicating biofilms of multiple human pathogens through
cell lysis.
Chopra and co-workers have reported biofilm-eradicating
dicationic porphyrins, XF-70 and XF-73, to have potent
MBEC values against S. aureus SH1000 at 2 µg/mL (2.62
µM) compared to MBEC >256 µg/mL for a panel of diverse
antibiotic controls (Fig. 3b) [55,56]. Kill kinetic experiments
were also conducted for XF-70 and XF-73 against stationary
phase S. aureus and compared to a panel of available antimi-
crobial agents. Although a slight decrease in killing rates
against stationary phase cultures with respect to exponential
4 Current Topics in Medici nal Chemistry, 2017, Vol. 17, No. 14 Garrison and Huigens III
phase cultures was observed, both compounds reported
eradication of the persistent cultures to below limits of detec-
tion (~7 log-fold reduction in CFU/mL) after 270 minutes of
treatment. The MIC:MBEC ratio for XF-70 and XF-73 is 2
(MIC 1 µg/mL; MBEC 2 µg/mL) demonstrating these agents
are equally as active effective against S. aureus planktonic
and biofilm cells.
Fig. (3). a) Barrel-stave model of AMP-promoted bacterial cell
death, which is one proposed mechanism for polycationic antibacte-
rial agents. b) S tructures of A MP-inspired cationic biofilm-
eradicating agents
Although reduction in membrane integrity was observed
with XF-70 and XF-73, the killing of S. aureus was not de-
termined to be the result of membrane lysis. Rather, an inter-
action with cellular membranes disrupts membrane potential
resulting in release of potassium ions and ATP in addition to
inhibition of macromolecular synthesis. At the time of this
review, Destiny Pharma has completed four phase I/IIa clini-
cal studies with XF-73 in the UK and Europe [57].
2.2. ADEP 4-Rifampicin Combination Strategy
Acyldepsipeptides (ADEPs) are antibiotics isolated from
Strepomyces hawaiiensis that exhibit potent activities against
Gram-positive bacteria. Brötz-Oesterhelt and co-workers
reported impressive antibacterial results for ADEP 1 and
related synthetic analogues against several drug-resistant
human pathogens, including methicillin-resistant Staphylo-
coccus aureus (MRSA) with IC50 values of 8.76 µM (6.3
µg/mL) for the natural product ADEP 1, 0.50 µM (0.4
µg/mL) and 64.9 nM (0.05 µg/mL) for the synthetic ana-
logues ADEP 2 and ADEP 4, respectively (Fig. 4a) [58].
Furthermore, ADEP 1 and ADEP 4 show efficacy using in
vivo models against S. aureus and Enterococcus faecalis
with ADEP 4 demonstrating the ability to rescue 80% of
mice from fatal S. aureus infections following a single 12.5
mg/kg dose. Through genomic analyses, the target for ADEP
compounds was identified as ClpP (caseinolytic protease), a
highly conserved bacterial peptidase [58].
The ClpP protease system is an energy-dependent proc-
ess that is responsible for important biological functions such
as protein quality control and the removal of protein debris
following bacterial stress events [59]. Under normal physio-
logical conditions, ClpP proteases require binding to AT-
Pases which unfold proteins and translocate them into the
ClpP proteolytic sites. Crystal structures of ADEP 1 bound
to Escherichia coli ClpP reveal that these antibacterial agents
occupy an area of the ATPase binding site [60]. This interac-
tion promotes the conversion of the ClpP entrance pore from
a closed- to an open-gate form (Fig. 4b). This conformation
bypasses the requirement for an associated ATPase and per-
mits the unregulated entrance of unfolded proteins into the
degradation chamber. The concomitant increase of ClpP ac-
tivity redirects proteolysis from native physiological sub-
strates to nascent peptides, resulting in inhibition of cell divi-
sion and, ultimately, cell death.
In contrast with previous studies wherein the evaluation
of ADEP-promoted protein degradation was performed with
short exposure times and against rapidly-growing cells,
Lewis and co-workers showed via proteomic analysis that
exposure of stationary phase MRSA to ADEP 4 resulted in a
reduced abundance of 24% of cellular proteins compared to
an untreated control [42]. This is especially useful as station-
ary phase populations of S. aureus are typically non-dividing
and polypeptide synthesis therein is highly downregulated
[61]. This finding also suggested that the growth-independent
mechanism of action could allow for the eradication of dor-
mant persister cells. Thus, ADEP 4 was evaluated against a
population of persister cells and it was found that this agent
eradicated persister cells to the limit of detection. Con-
versely, rifampicin demonstrated no effect on the viability of
these persister populations.
In addition, ADEP 4 demonstrated excellent killing of
stationary phase S. aureus after 2 days treatment, reporting a
reduction of viable cell counts by 4 log10 [42]. However,
since ClpP is non-essential to S. aureus and null clpP muta-
tion frequency is high ( ~10-6 ), population rebounds were
observed after day 3 in these experiments. In attempts to
Eradicating Bacterial Biofilms with Natural Products Current Topics in Medici nal Chemistry, 2017, Vol. 1 7, No. 14 5
suppress resistant mutants, stationary phase S. aureus popu-
lations w ere co-treated with ADEP 4 and conventional anti-
biotics. The pairing of ADEP 4 with rifampicin resulted in
complete eradication of the persistent cells to the limit of
detection without any observable rebound in bacterial popu-
lation. This result was initially surprising as clpP mutation
should confer resistance to a detectable population of cells.
In a subsequent experiment, it was determined that although
treatment of a clpP mutant strain with the conventional anti-
biotics linezolid and rifampicin demonstrated similar MIC
values to those of the wild-type, the clpP mutant strains were
less able to produce persister populations by an order of 10-
to 100-fold concluding that mutations in clpP (e.g., those
that may result from ADEP 4 treatment) render the bacteria
less fit and thus, more susceptible to certain antibiotics.
The ability of this ADEP 4-rifampicin combination strat-
egy to eradicate bacterial biofilms was also evaluated in 96-
well plates (non-Calgary Biofilm Device) [42]. Following a
24-hour establishment of S. aureus UAMS-1 biofilms, wells
were treated with the ADEP 4-rifampicin combination re-
sulting in eradicated biofilms to the limit of detection (>4
log-fold reduction) following 3 days of treatment (ADEP 4
at 6.49 µM, 5 µg/mL; 10 X MIC; rifampicin at 0.49 µM, 0.4
µg/mL; 10 X MIC in this combination). ADEP 4-rifampicin
was also shown to be highly efficacious in a deep-seated
mouse thigh infection model. After 24-hour infection devel-
opment in mice, histopathology was performed to confirm
the adherence of bacterial biofilms to thigh muscle tissue.
Treatment of the deep-seated infection with ADEP 4-
rifampicin resulted in an eradication of the biofilm-
associated infection to the limit of detection within 24 hours
(>4 log-fold reduction), whereas rifampicin, vancomycin or
a combination of both could decrease viable cell counts, but
not eradicate the biofilm infection.
Recently, Sello and co-workers disclosed an enhance-
ment of ADEP analogues via introduction of conformational
restriction by replacement of key residues in the macrocyclic
core [62]. There was a 20-fold improvement of in vitro S.
aureus activity from MIC = 649 nM (0.5 µg/mL; from ref
[42]) for ADEP 4 compared to 30.6 nM (0.024 µg/mL) for
their novel lead, ADEP “1g”. Although no biofilm eradica-
tion or in vivo assays were conducted during this study, these
developments demonstrate that the ADEP-rifampicin combi-
nation therapy approach is still ripe for development.
2.3. Mitomycin C
Mitomycin C is a natural product isolated from Strepto-
myces caespitosus endowed with a reactive aziridine hetero-
cycle critical for its anticancer and antibacterial activities
[63,64]. Mitomycin C, an FDA-approved anticancer agent,
has recently exhibited an ability to eradicate persistent bacte-
rial populations [65,66]. Following chemical or enzymatic
Fig. (4). a) Structures of the natural product, ADEP 1, and its synthetic analogues. b ) Binding of ADEP to the ClpP protease promotes open-
ing of the central pore and allows entry of unfolded proteins into proteolytic sites. Dysregulated ClpP indiscriminately degrades polypep-
tides, resulting in self-digestion and cell death.
6 Current Topics in Medici nal Chemistry, 2017, Vol. 17, No. 14 Garrison and Huigens III
reduction of the electron-poor quinone moiety to an electron-
rich hydroquinone, a spontaneous cascade reaction occurs
wherein the aziridine ring opens to form an unstable viny-
logous quinone methide (Fig. 5). This reactive quinone me-
thide motif serves as an electrophilic site which undergoes
nucleophilic attack by a DNA base (commonly from the N2
amino group of a guanine residue). This adduct forms a sec-
ond alkylating site via the elimination of the adjacent carba-
mate followed by subsequent nucleophilic attack by a second
guanine residue on an opposing DNA strand that results in
covalent cross-linking and cellular d eath.
Fig. (5). Mitomycin C undergoes reduction, triggering a spontane-
ous cascade reaction that results in the cross-linking of DNA.
Bacterial cytoplasm is an inherently reductive medium,
thus Wood and co-workers surmised that the passive diffu-
sion and spontaneous DNA cross-linking by mitomycin C
could be an effective strategy for the eradication of non- or
slow-growing persistent populations [65,66]. Indeed, treat-
ment of a panel of pathogens (Escherichia coli, Pseudo-
monas aeruginosa, and S. aureus) with mitomycin C re-
vealed a propensity of this drug to eradicate stationary phase
cells with high efficacy at 29.9 µM (10 µg/mL; >7 log-fold
reduction of CFU/mL). Using in vitro assays against S.
aureus biofilms, mitomycin C eradicated biofilms beyond
the limit of detection at 29.9 µM (non-Calgary Biofilm De-
vice or 96-well plate experiments), whereas ciprofloxacin
yielded 18 ± 2% biofilm survival rates at 15 µM (5 µg/mL).
During these investigations, an in vitro Lubbock chronic
wound biofilm model [67] was also used to simulate growth
conditions of clinical biofilm-afflicted wounds, which differ
from more generally used lab conditions. In these experi-
ments, mitomycin C eradicated S. aureus biofilms to beyond
the limit of detection (>7 log-fold reduction) at 29.9 µM
[65]. Conversely, control antibiotics ciprofloxacin and am-
picillin had only a minimal effect on bacterial viability in
these assays. In vivo assessment of mitomycin C proved ef-
fective when using a nematode Caenorhabditis elegans En-
terohaemorrhagic Escherichia coli (EHEC) infection model.
Nematodes were fed on lawns of EHEC, a pathogenic strain
of E. coli, for two days prior to drug treatment to promote a
fully established infection. Following drug administration,
the control antibiotics ciprofloxacin and ampicillin improved
survival rates (~60-64%) with respect to uninfected controls
after 12 days; however, the rates of mitomycin C were much
higher (~83%), likely due to the ability of mitomycin C to
eradicate the persister cells responsible for the re-
establishment of infection [48].
Using single-gene deletion strains of E. coli, the activity
of mitomycin C was evaluated against mutants lacking uvrA,
uvrB and uvrC genes [65]. These genes, forming the
UvrABC complex, serve to repair DNA cross-links in E. coli
[68]. Against these knockout strains, mitomycin C eradicated
bacterial cultures to beyond the limit of detection within 30
minutes of treatment (>7 log-fold reduction of all knockout
strains), confirming that the anti-persister activity was a re-
sult of DNA cross-linking.
Lewis and co-workers discovered mitomycin C to be ac-
tive against Borrelia burgdorferi, the causative agent of
Lyme disease [69]. Therein, mitomycin C was found to
eradicate late-exponential phase B. burgdorferi cells within
24 hours of treatment at 4.8 µM (1.6 µg/mL, 8 X MIC). Sur-
prisingly, it was shown that stationary phase cultures of B.
burgdorferi were more susceptible to mitomycin C, suffering
complete eradication after 24-hours at 2.4 µM (0.8 µg/mL).
In both cases, treatment with mitomycin C resulted in no
observable populations of persister cells. This finding was
especially significant as recalcitran ce B. burgdorferi infec-
tions are likely due to the growth of drug-tolerant persisters
rather than drug-resistant mutants.
There are, however, known toxicity concerns that could
prove to be a hindrance for the use of mitomycin C as an
antibacterial agent. Known clinical toxicities for mitomycin
C administration include: bone marrow suppression, renal
Eradicating Bacterial Biofilms with Natural Products Current Topics in Medici nal Chemistry, 2017, Vol. 1 7, No. 14 7
failure, and hemolytic anemia [70].
During Wood’s investi-
gations, the concentrations reported to combat bacterial per-
sister cells (29.9-44.9 µM; 10-15 µg/mL) are significantly
higher than what is appropriate for intravenous cancer treat-
ments (1.5-6.0 µM; 0.5-2.0 µg/mL) [71]. However, Wood
and co-workers report that the effective concentration of
mitomycin C as an antibacterial agent should be much lower
in practice. Additionally, Shields and co-workers have re-
ported on the safe topical administration of mitomycin C at
up to 2.59 mM (400 µg/mL) [72]. With prior FDA-approval
and a well-established toxicity profile, mitomycin C could be
applicable to clinical treatment of persister-cell- and biofilm-
associated bacterial infections.
2.4. Phenazine Antibiotic Inspired Small Molecules
Phenazine antibiotics are a class of redox-active secon-
dary metabolites mainly produced by Streptomyces and
Pseudomonas species. Huigens and co-workers became in-
terested in phenazine antibiotics due to the ability of P.
aeruginosa to co-infect and clear established S. aureus infec-
tions in cystic fibrosis patients [73]. This competitiv e advan-
tage is attributed to P. aeruginosa using phenazine antibiot-
ics (i.e., pyocyanin) to target S. aureus [74-76] and it was
hypothesized that these established infections were biofilm
in nature, and thus, phenazine antibiotic inspired small mole-
cules could possess the ability to eradicate S. aureus (and
other pathogen) biofilms. This phenazine antibiotic inspired
inter-bacterial warfare strategy has led to a chemically di-
verse series of novel biofilm-eradicating agents.
During initial studies, a small panel of 13 phenazine anti-
biotics and synthetic phenazines were synthesized and
screened for antibacterial activity against S. aureus and S.
epidermidis. Interestingly, the marine phenazine antibiotic,
1-hydroxy-2-bromophenazine (HP-1; MIC = 6.25 µM), an d
a related halogenated phenazine analogue (HP-2; MIC =
1.56 µM) demonstrated potent antibacterial activities against
S. aureus and S. epidermidis (Fig. 6a) [77]. Following this
initial find, HP-2 (MBEC = 150 µM) and HP-3 (MBEC =
81.3 µM) demonstrated effective eradication of methicillin-
resistant S. aureus (MRSA clinical isolate) biofilms using
non-Calgary Biofilm Device 96-well plate assays [41].
Through the development of a more chemically diverse
library of HP analogues, HP-4 proved to be the most potent
biofilm-eradicator against MRSA BAA-1707 (MBEC = 6.25
µM), methicillin-resistant Staphylococcus epidermidis 35984
(MRSE 35984; MBEC = 2.35 µM), and vancomycin-
resistant Enterococcus faecium 700221 (VRE; MBEC = 0.20
µM) using Calgary Biofilm Device assays [8,43]. In addition,
HP-5, HP-6 and HP-7 exhibited remarkable biofilm-
eradication activities (Fig. 6b). Conversely, vancomycin
reported MBECs of >2000 µM against both MRSA and
MRSE and 150 µM against VRE in these Calgary Biofilm
Device assays. Interestingly, HP-7 demonstrated planktonic
activity (MIC = 3.13 µM) against Mycobacterium tuberculo-
sis H37Ra, a bacterial strain which is well known for its per-
sistent nature. In time-dependent kill kinetics assays against
MRSA-2 stationary cultures, HP-4 was able to slowly re-
duce viable cell counts by >3 log-fold at 12.5 µM (corre-
sponding MBEC value against MRSA-2) after 24 hours
while control antibiotics vancomycin and linezolid reduced
cell counts by <1 log fold at 100 µM. This effect is not due
to antibiotic resistance as MRSA-2 is susceptible to both
vancomycin and linezolid in MIC assays. This persister kill-
ing effect was dramatically different than membrane-active
QAC-10, which rapidly killed MRSA-2 stationary popula-
tions in these experimen ts at 100 µM [43].
The antibacterial activities of phenazine antibiotics have
been attributed to their redox potential [75,78]. The central
ring of the phenazine heterocycle can undergo redox cycling
to generate superoxide anions. During inv estigations into the
mode of action of HP small molecules, however, we ob-
served minimal effects on HP activity when bacteria were
co-treated with known superoxide quenching agents or free
radical scavengers (e.g., tiron, thiourea, manganese(III),
tetrakis(4-benzoic acid)porphyrin, ascorbic acid) [8]. Inter-
estingly, iron(II) and copper(II) co-treatment reduced th e
antibacterial activities of active HPs in MIC assays. It was
also determined that HP-2 exhibited a strong affinity toward
iron(II)- and copper(II)-cations, likely through chelation of
the phenolic hydroxyl and adjacent nitrogen atom of the H P
scaffold, resulting in the formation of 2:1 HP:metal com-
plexes. Together, these data suggest that HPs do not elicit
bacterial death primarily through redox activity but rather, a
unique metal(II)-dependent mechanism. Interestingly,
phenazine antibiotic 1-hydroxyphenazine was recently re-
ported to exhibit antifungal activity through an iron-
starvation mechanism via the chelation of the phenolic oxy-
gen and adjacent nitrogen atoms to iron(III) in a 2:1
phenazine:metal complex resulting in iron-starvation [79].
Based on the structural similarities (i.e., the retention of
the metal chelation site) to HPs, the Huigens lab expanded
their biofilm eradication program to include halogenated
quinolines (HQs) through a scaffold-hopping approach
[80,81]. Results from preliminary MIC assays against plank-
tonic cells were promising, with HQ-1 reporting MICs of
0.39 µM against S. aureus and 1.17 µM against S. epider-
midis. Using various synthetic approaches and subsequent
Calgary Biofilm Device assays, several potent biofilm-
eradicating HQs have been identified [82,83].
Initially, HQ-1 demonstrated effective biofilm eradica-
tion activities against MRSA (MBEC = 188 µM), MRSE
(93.8 µM), and VRE (1.5 µM). Lead optimization via syn-
thetic tuning of the 2-position of the quinoline heterocycle
yielded several reductive amination and alkylated analogues
with improved biofilm-eradication activities [83]. HQ-3
proved to be the most potent biofilm eradicator against
MRSE, reporting an MBEC of 3.0 µM. Against VRE, HQ-4
reported an MBEC of 1.0 µM during this study.
As was the case with HPs, co-treatment with metal(II)
cations modulates the activities of HQs, suggesting that these
biofilm-eradicating small molecules act through a related
mechanism(s); however, a more precise elucidation of the
HP and HQ mechanism(s) of action is underway and prom-
ises to shed new light on biofilm viability. The HP/HQ scaf-
fold is promising for potential therapeutic use as demon-
strated by the low cytotoxicity against HeLa cells, lysis of
red blood cells and animal toxicity that has been observed
during preliminary investigations [8,43,82,83].
8 Current Topics in Medici nal Chemistry, 2017, Vol. 17, No. 14 Garrison and Huigens III
Fig. (6). a) Discovery and progression of biofilm-eradicating halogenated phenazines and halogenated quinolines. b) Structure-activity rela-
tionships for lead biofilm-eradicating agents against MRSA, MRSE and VRE.
CONCLUSION
Although we have had success in the discovery of thera-
peutically useful antibiotics to treat acute bacterial infections
in humans, current antibiotic therapies have shown little ef-
fectiveness against persistent biofilm-associated infections.
Despite this, a small yet diverse collection of natural prod-
ucts and natural product-inspired compounds has emerged
that operate through unique, growth-independent modes of
action leading to the eradication of persistent bacterial
Eradicating Bacterial Biofilms with Natural Products Current Topics in Medici nal Chemistry, 2017, Vol. 1 7, No. 14 9
biofilms. This unique collection of promising natural prod-
ucts and inspired analogues will continue to inspire efforts
aimed at harnessing microbial warfare agents and strategies
to effectively treat chronic and recurring bacterial infections
that arise from persistent bacterial biofilms.
LIST OF ABBR EVIATIONS
AMP = Antimicrobial peptide
ATP = Adenosine triphosphate
CBD = Calgary biofilm device
DNA = Deoxyribonucleic acid
HP = Halogenated phenazine
HQ = Halogenated quinoline
MIC = Minimum inhibitory concentration
MBC = Minimum bactericidal concentration
MBEC = Minimum biofilm eradication concentration
µM = Micromolar
CONFLICT OF INTEREST
The author(s) confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
We would like to acknowledge the University of Florida
for start-up funds and seed funding to support our biofilm
eradication discovery platforms. We would also like to thank
Akash Basak for providing useful comments in the prepara-
tion of this manuscript.
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