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Eradicating Bacterial Biofilms with Natural Products and Their Inspired Analogues that Operate Through Unique Mechanisms

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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 inhibitors of bacterial growth, so it should be little surprise that our arsenal of growth-inhibiting agents have 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 bacterial 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 recurring bacterial infections due to persistent bacterial biofilms.
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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.
REFERENCES
[1] Anon. A Nobel endeavor. Nat. Rev. Microbiol., 2010, 8, 755.
[2] Kaufmann, S. H. E. Robert Koch, the Nobel Prize, and the Ongoing
Threat of Tub erculosis. N. Eng. J. Med. 2005, 353, 2423-2426.
[3] Baxter, A. G. SCIENCE AND SOCIETY: Louis Pasteur’s beer of
revenge. Nat. Rev. Immunol. 2001, 1, 229-232.
[4] Bennett, J. W.; K ing-Thom, C. Alexander Fleming and the discov-
ery of penicillin. Adv. Appl. Microbiol. 2001, 49, 163-184.
[5] Aminov, R. I. A Brief History of the Antibiotic Era: Lessons
Learned and Challenges for the Future. Front. Microbiol. 2010, 1,
134.
[6] Lewis, K. Platforms for antibiotic discovery. Nat. Rev. Drug Dis-
cov. 2013, 12, 371-387.
[7] Pawlowski, A. C. ; Johnson , J. W.; Wright, G. D. Evolvin g medici-
nal chemistry strategies in antibiotic discovery. Curr. Opin. Bio-
technol. 2016, 42, 108-117.
[8] Garrison, A. T.; Abouelhassan, Y.; Norwood IV, V. M.; Kallifidas,
D.; Bai, F.; Nguyen, M.; Rolfe, M., Burch, G. M.; Jin, S.; Luesch,
H.; Huigens III, R. W. Structure-Activity Relationships of a Di-
verse Class of Halogenated Phenazines that Targets Persistent, An-
tibiotic-Tolerant Bacterial Biofilms and Mycobacteriu m tuberculo-
sis. J. Med. Chem. 2016, 59, 3808-3825.
[9] Miller, M. B. ; Bassler, B. L. Qu orum sensin g in bacteria. Ann. Rev.
Microb iol. 2001, 55, 165-199.
[10] Donlan, R. M.; Costerton, J. W. Biofilms: Survival Mechanisms of
Clinically Relevant Microorganisms. Clin. Microbiol. Rev. 2002,
15, 167-193.
[11] Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Bacterial
Biofilms: From the Natural Environment to Infectious Diseases.”
Nat. Rev. Microbiol. 2004, 2, 95-108.
[12] Lewis, K. Persister cells, dormancy and infectious disease. Nat.
Rev. Microbiol. 2007, 5, 48-56.
[13] Wood, T. K. Combatting Bacterial Persister Cells. Biotechnol.
Bioengineer. 2016, 113, 476-483.
[14] Conlon, B. P. Staphylococcus aureus chronic and relapsing infec-
tions: Evidence of a role for persister cells. BioEssays 2014, 36,
991-996.
[15] Balaban, N. Q.; Merrin, J.; Chait, R.; Kowalik, L.; Leiber, S.; Bac-
terial persisten ce as a phenotype switch. Science 2004, 305, 1622-
1625.
[16] Wood, T. K.; Knabel, S. J. ; Kwan, B. W. Bacterial persister cell
formation and dormancy. Appl. Environ. Microbiol. 2013, 79 ,
7116-7121.
[17] Webb, J. S.; Givskov, M.; Kjelleberg, S. Bacterial Biofilms: pro-
karyotic adventures in multicellularity. Curr. Opin. Microbiol.
2003, 6, 578-585.
[18] Archer, N. K.; Mazaits, M. J.; Costerton , J. W.; Leid, J. G .; Powers,
M. E.; Shirtliff, M. E. Staphylococcus aureus biofilms: properties,
regulation and roles in human disease. Virulence 2011 , 2, 1-15.
[19] Harbarth, S.; Theuretzbacher, U.; Hackett, J. Antibiotic research
and development: Business as usual? J. Antimicrob. Chemother.
2015, 70, 1604-1607.
[20] Musk, D. J.; Hergenrother, P. J. Chemical countermeasures for the
control of bacterial biofilms: Effective compounds and promising
targets. Curr. Med. Chem. 2006, 13, 2163-2177.
[21] Wolcott, R.; Dowd, S. The role of biofilms: Are we hitting the right
target? Plast. Reconstr. Surg. 2011, 127, Suppl 1:28S-35S.
[22] Otto, M. Staphylococcal Biofilms. Curr. Top. Microbiol. Immunol.
2008, 322, 207-228.
[23] Paharik, A. E.; Horswill, A. R. The Staphylococcal Biofilm: Ad-
hesins, Regulation, and Host Response. Microbiol. Spectr. 2016,
4(2).
[24] Gupta, P.; Sarkar, S.; Das, B.; Bhattacharjee, S.; Tribedi, P.
Biofilm, pathogenesis and preventiona journey to break the wall:
a review. Arch. Microbiol. 2016, 198, 1-15.
[25] Halstead, F. D.; Rauf, M.; Moiemen, N. S.; Bamford, A.; Wearn,
C. M.; Fraise, A. P.; Lund, P. A.; Oppenheim, B. A.; Webber, M.
A. The Antibacterial Activity of Acetic Acid against Biofilm-
Producing Pathogens of Relevance to Burns Patients. PLoS One
2015, 10, pp 15.
[26] Phillips, P. L.; Yang, Q.; Schultz, G. S. The effect of neg ative
pressure wound therapy with periodic instillation using antimicro-
bial solutions on Pseudomonas aeruginosa biofilm on porcine skin
explants. Int. Wound J. 2013, 10, 48-55.
[27] Ganesh, K.; Sinha, M.; Mathwe-Steiner, S. S.; Das, A.; Roy, S.;
Sen, C. K. Chronic Wound Biofilm Model. Adv. Wound Care 2015,
4, 382-388.
[28] Peel, T. N. ; Buising, K. L.; Choong, P. F. Diagnosis and manage-
ment of prosthetic joint infection. Curr. Opin. Infect. Dis. 2012, 25,
670-676.
[29] Fernández, J.; Greenwood-Quaintance, K. E.; Patel, R. In vitro
activity of dalbavancin against biofilms of staphylococci isolated
from prosthetic joint infections. Diagn. Microbiol. Infect. Dis. 2016
in press.
[30] Kim, M. K.; Drescher, K.; Pak, O. S.; Bassler, B. L.; Stone, H. A.
Filaments in curved streamlines: Rapid formation of Staphyloco c-
cus aureus biofilm streamers. New J. Phys. 2014, 16, 065024.
[31] Heim, C. E.; Hanke, M. L.; K ielian, T. A mouse model of Staphy-
lococcus catheter-associated biofilm infection. Methods Mol. Biol.
2014, 1106, 183-191.
[32] Percival, S. L.; Suleman, L.; Vuotto, C.; Donelli, G. Healthcare-
associated infections, medical devices and biofilms: risk, tolerance
and control. J. Med. Microbiol. 2015, 64, 323-334.
[33] Chauhan, A.; Ghigo, J. M.; Beloin, C. Study of in vitro catheter
biofilm infections using pediatric central venous catheter implanted
in rat. Nat. Protoc. 2016, 11, 525-541.
[34] Fux, C. A.; Quigley, M.; Worel, A. M.; Post, C.; Zimmerli, S.;
Ehrlich, G; Veeh, R. H. Biofilm-related infections of cerebrospinal
fluid shunts. Clin. Microbiol. Infect. 2006, 12, 331-337.
[35] Weisser, M.; Schoenfelder, S. M. K., Osasch, C.; Arber, C.; Grat-
wohl, A.; Frei, R., Eckart, M.; Flückiger, U.; Ziebuhr, W. Hyper-
variability of Biofilm Formation and Oxacillin Resistance in a
Staphylococcus Strain Causing Persistent Severe Infection in an
Immunocompromised Patient. J. Clin. Microbiol. 2010, 48, 2407-
2412.
10 Current Topics in Medicinal Chemistry, 2017, Vol. 17, No. 14 Garrison and Huigens III
[36] Marsh, P. D. Dental plaque as a biofilm: the significance of pH in
health and caries. Compend. Contin. Educ. Dent. 2009, 30, 76-78.
[37] Worthington, R. J.; Richards, J. J.; Melander, C. Small molecule
control of bacterial biofilms. Org. Biomol. Chem. 2012, 10, 7457-
7474.
[38] Rabin, N.; Zheng, Y.; Opoku-Temeng, C.; Du, Y.; Bonsu, E.; Sin-
tim, H. O. Biofilm formation mechanisms and targets for develop-
ing antibiofilm agents. Future Med. Chem. 2015, 7, 493-512.
[39] Ceri, H.; Olson, M. E.; Stremick, C.; Read, R. R.; Morck, D.; Bu-
ret, A. The Calgary Biofilm Device: New Technology for Rapid
Determination of Antibiotic Susceptibilities of Bacterial Biofilms.
J. Clin. Microbiol. 1999, 37, 1771-1776.
[40] Jennings, M. C.; Ator, L. E.; Paniak, T. J.; Minbole, K. P. C.;
Wuest, W. M. Biofilm eradicating properties of quaternary ammo-
nium amphiphiles: Simple mimics of antimicrobial peptides.
ChemBioChem 2014, 15, 2211-2215.
[41] Garrison, A. T.; Bai, F.; Abouelhassan, Y., Paciaroni, N. G.; Jin, S.;
Huigens III, R. W. Bromophenazine derivatives with potent inhibi-
tion, dispersion and eradication activities against Staphylococcus
aureus biofilms. RSC Adv. 2015, 5, 1120-1124.
[42] Conlon, B. P.; Nakayasu, E. S.; Fleck, L. E.; LaFleur, M. D.;
Isabella, V. M.; Coleman, K.; Leonard, S. N.; Smith, R. D.; Ad-
kins, J. N.; Lewis, K. Activated ClpP kills persisters and eradicates
a chronic biofilm infection. Nature 2013, 503, 365-370.
[43] Garrison, A. T.; Abouelhassan, Y.; Kallifidas, D.; Bai, F.; Uk-
hanova, M.; Mai, V.; Jin, S.; Luesch, H.; Huigens III, R. W. Halo-
genated Phenazines that Potently Eradicate Biofilms, MRSA Per-
sister Cells in Non-Biofilm Cultures, and Mycobacterium tubercu-
losis. Angew. Chemie Int. Ed. 2015, 54, 14819-14823.
[44] Keren, I.; Kaldalu, N.; Spoering, A.; Wang, Y.; Lewis, K. Persister
cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 2004,
230, 13-18.
[45] Lechner, S.; Lewis, K.; Bertram, R. Staphylococcus aureus persis-
ters tolerant to bactericidal antibio tics. J. Mol. Microbiol. Biotech-
nol. 2012, 22, 235-244.
[46] Brogden, K. A. Antimicrobial peptides: pore formers or metabolic
inhibitors in bacteria? Nat. Rev. Microbio. 2005, 3, 238-250.
[47] Zasloff, M. Antimicrobial peptides of multicellular organisms.
Nature 2002, 415, 389-395.
[48] Wieprecht, T.; Apostolov, O.; Beyermann, M.; and Seelig, J. De-
tergent-like actio n of the antibiotic peptide surfactin on lipid mem-
branes. Biochemistry 2003, 39, 442-452.
[49] Findlay, B.; Zhanel, G. G.; Schweizer, F. Cationic amphiphiles, a
new generation of antimicrobials inspired by the natural antimicro-
bial peptide scaffold. Antimicrob. Agents Chemother. 2010, 54,
4049-4058.
[50] Brogden, K. A.; De Lucca, A. J.; Bland, J.; Elliott, S. Isolation of
an ovine pulmonary surfactant-associated anionic peptide bacteri-
cidal for Pasteurella haemolytica. Proc. Natl Acad. Sci. USA 1996,
93, 412-416.
[51] Brotz, H.; Bierbaum, G.; Leopold, K.; Reynolds, P. E.; Sahl, H. G.
The lantibiotic mersacidin inhibits peptidoglycan synthesis by tar-
geting lipid II. Antimicrob. Agents Chemother. 1998, 42, 154-160.
[52] Park, C. B.; K im, H. S.; Kim, S. C. Mechanism of action of the
antimicrobial peptide buforin II: buforin II kills microorganisms by
penetrating the cell membrane and inhibiting cellular functions.
Biochem. Biop hys. Res. Commun. 1998, 244, 253 -257.
[53] Patrzykat, A.; Friedrich, C. L.; Zhang, L.; Mendoza, V.; Hancock,
R. E. Sublethal concentrations of pleurocidin derived antimicrobial
peptides inhibit macromolecular synthesis in Escherichia coli. An-
timicrob. Agents Chemother. 2002, 46, 605-614.
[54] Mitchell, M. A.; Iannetta, A. A.; Jennings, M. C.; Fletcher, M. H.;
Wuest, W. M.; Minbiole, K. P. Scaffold-Hopping of Multicationic
Amphiphiles Yields Three New Classes of Antimicrobials.
ChemBioChem 2015, 16, 2299-2303.
[55] Ooi, N.; Miller, K.; Hobbs, J.; Rhys-Williams, W.; Love, W.;
Chopra, I. XF-73, a novel antistaphylococcal membrane-active
agent with rapid bactericidal activity. J. Antimicrob. Chemother.
2009, 64, 735-740.
[56] Ooi, N.; Miller, K.; Randall, C.; Rhys-Williams, W.; Love, W.;
Chopra, I. J. Antimicrob. Chemother. 2010, 65, 72-78.
[57] Destiny Pharma Ltd. www.destinypharma.com/news_current.shtml
(Accessed June 15, 2016).
[58] Brötz-Oesterhelt, H; Beyer, D.; Kroll, H. P.; Endermann, R.; Ladel,
C.; Schroeder, W.; Hinzen, B.; Raddatz, S.; Paulsen, H .; Hen-
ninger, K.; Bandow, J. E.; Sahl, H. G.; Labischinski, H. Dysregula-
tion of bacterial proteolytic machinery by a new class of antibiot-
ics. Nat. Med. 2005, 11, 1082-1087.
[59] Lee, B. G.; Park, E. Y. ; Lee, K. E.; Jeon, H.; Sung, K. H.; Paulsen,
H.; Rübsamen-Schaeff, H.; Brötz-Oesterhelt, H.; Song, H. K.
Structures of ClpP in complex with acy ldepsipeptide antibiotics re-
veal its activation mechanism. Nat. Struc. Mol. Bio. 2010, 17, 471-
478.
[60] Li, D. H.; Chung, Y. S.; Gloyd, M.; Joseph, E.; Ghirlando, R.;
Wright, G. D.; Cheng, Y. Q.; Maurizi, M. R.; Guarné, A.; Ortega,
J. Acyldepsipeptide Antibiotics Induce the Formation of a Struc-
tured Axial Channel in ClpP: A Model for the ClpX/ClpA-Bound
State of ClpP. Chem. Biol. 2010, 17, 959-969.
[61] Michalik, S; Liebeke, M.; hlke, D.; Lalk, M.; Bernhardt, J.;
Gerth, U.; Hecker, M. Proteolysis during long-term g lucose starva-
tion in Staphylococcus aure us COL. Proteomics 2009, 9, 4468-
4477.
[62] Carney, D. W.; Schmitz, K. R.; Truong, J. V.; Sauer, R. T.; Sello, J.
K. Restriction of the Conformational Dynamics of the Cyclic
Acyldepsipeptide Antibiotics Improves Their Antibacterial Activ-
ity. J. Am. Chem. Soc. 2014, 136, 1922-1929.
[63] Danshiitsoodol, N.; de Pinho, C. A.; Matoba, Y.; Kumagai, T.;
Sugiyama, M. The mitomycin C (MMC)-binding protein from
MMC-producing microorganisms protects from the lethal effect of
bleomycin: crystallographic analysis to elucidate the binding mode
of the antibiotic to the protein. J. Mol. Biol. 2006, 360, 398-408.
[64] Tomasz, M. Mitomycin C: small, fast and deadly (but very selec-
tive). Chem Biol. 1995, 2, 575-579.
[65] Kwan, B. W.; Chowdhury, N.; Wood, T. K. Combatting bacterial
infections by killing persister cells with mitomycin C. Environ. Mi-
crobiol. 2015, 17, 4406-4414.
[66] Szybalski, W.; Iyer, V. N . Crosslinking of DNA by enzymatically
or chemically activated mitomycins and profiromycins, bifunction-
ally ‘alkylating’ antibiotics. Fed. Proc. 1964, 23, 946-957.
[67] Su n, Y.; Dowd, S. E.; Smith, E.; Rhoads, D. D.; Wolcott, R. D. In
vitro multispecies Lubbock chronic wound biofilm model. Woun d
Repair Regen. 2008, 16, 805-813.
[68] Weng, M.-W.; Zheng, Y.; Jasti, V. P.; Champeil, E.; Tomasz, M.;
Wang, Y.; Basu, A. K.; Tang, M. S. Repair of mitomycin C mono-
and interstrand cross-linked DNA adducts by UvrABC: a new
model. Nucleic Acids Res. 2010, 38, 6976-6984.
[69] Sh arma, B.; Brown, A. V.; Matluck, N. E.; Hu, L. T.; Lewis, K.
Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms
Drug-Tolerant Persister Cells. Antimicrob Agents Chemother.
2015, 59, 4616-4624.
[70] Doll, D. C.; Weiss, R. B.; Issell, B. F. Mitomycin: ten years after
approval for marketing. J. Clin. Oncol. 1985, 276-286.
[71] Bradner, W.T. Mitomycin C: a clinical update. Cancer Treat. Rev.
2001, 27, 35-50.
[72] Sh ields, C. L.; Naseripo ur, M.; Shields, J. A. Topical mitomycin C
for extensive, recurrent conjunctival-corneal squamous cell carci-
noma. Am. J. Ophthalmol. 2002, 133, 601-606.
[73] Saiman, L. Microbiology of early CF lung disease. Paediatr.
Respir. Rev. 2004, 5(Suppl A), S367-S369.
[74] Dietrich, L. E.; Okegbe, C.; Price-Whelan, A.; Sakhtah, H.; Hunter,
R. C.; Newman, D. K . Bacterial Community Morphogenesis Is In-
timately Linked to the Intracellular Redox State. J Bacteriol. 2013,
195, 1371-1380.
[75] Price-Whelan, A.; Dietrich, L. E.; Newman, D. K. Rethinking
‘secondary’ metabolism: physiological roles for phenazine antibiot-
ics. Nat. Ch em. Biol. 2006, 2, 71-78.
[76] Machan, Z. A.; Pitt, T. L.; White, W.; Watson, D.; Taylor, G. W.;
Cole, P. J.; Wilson, R. Interaction between Pseudomonas aerugi-
nosa and Staphylococcus aureus: description of an anti-
staphylococcal substance. J. Med. Microbiol. 1991, 34, 213-217.
[77] Borrero, N. V.; Bai, F; Perez, C.; Duong, B. Q.; Rocca, J. R.; Jin,
S.; Huige ns, R. W., III. Phenazine antibiotic inspired disco very of
potent bromophenazine antibacterial agents a gainst Staphylococcus
aureus and Staphylococcus epidermidis. Org. Biomol. Chem. 2014,
12, 881-886.
[78] Laursen, J. B.; Nielsen, J. Phenazine natural products: biosynthesis,
synthetic analogues, and biological activity. Chem. Rev. 2004, 104,
1663-1685.
[79] Briard, B.; Bomme, P.; Lechner, B. E.; Mislin, G. L. A .; Lair, V.;
Prevost, M. C.; Latgé , J. P.; Haas, H .; Beauvais, A. ́ Pseudomonas
aeruginosa manipulates redox and iron homeostasis of its microbi-
Eradicating Bacterial Biofilms with Natural Products Current Topics in Medici nal Chemistry, 2017, Vol. 1 7, No. 14 11
ota partner Aspergillus fumigatus via phenazines. Sci. Rep. 2015, 5,
8220.
[80] Abouelhassan, Y.; Garrison, A. T.; Burch, G. M.; Wong, W.; Nor-
wood, V. M., IV; Huigens, R. W., III. Discovery of quin oline small
molecules with potent dispersal activity against methicillin-
resistant Staphylococcus aureus and Staphylococcus epidermidis
biofilms using a scaffold hopping strategy. Bioorg. Med. Chem.
Lett. 2014, 24, 5076-5080.
[81] Abouelhassan, Y.; Garrison, A. T.; Bai, F.; Norwood, V. M., IV;
Nguyen, M.; Jin, S.; Huigens, R. W., III. A phytochemical-
halogenated quinoline combination therapy strategy for the treat-
ment of pathogenic bacteria. ChemMedChem 2015, 10, 1157-1162.
[82] Basak, A.; Abouelhassan, Y.; Huigens, R. W., III. Halogenated
quinolines discovered through reductive amination with potent
eradication activities against MRSA, MRSE and VRE biofilms.
Org. Biomol. Chem. 2015, 13, 10290-10294.
[83] Basak, A.; Abouelhassan, Y.; Norwood, V. M., IV; Bai, F.;
Nguyen, M.; Jin, S.; Huigens III, R. W. Synthetically Tuning the 2-
Position of Halogenated Quinolines: Optimizing Antibacterial and
Biofilm Eradication Activities via Alkylation and Reductive Ami-
nation Pathways. Chem. Eur. J. 2016, 22, 9181-9189.
DISCLAIMER: The above article has been published in Epu b (ahead of print) on the basis of the materials provided by the author. The
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PMID: 27966398
... Both MBDC and MBEC (minimal biofilm-eradication concentration) actually have the same nature. The latter is commonly used to characterize biofilm-eradication activity of antibacterials through MBC/MBEC ratios that for the biofilm-eradicating agents typically have values in the range of 1 to 3 (Garrison and Huigens, 2017). Considering the high levels of the MBC/ MBDC ratios observed here (about 2 to 3), DMNP can be viewed as a biofilm-eradicating agent ( Figure 7C). ...
... We compared the results of the MBC/MBDC measurements with those for the MBC/MBEC ratios for WT that were conducted with the MBEC Biofilm Inoculator (Innovotech, Canada) for DMNP and the conventional antibiotics (Table S3). Although DMNP exhibited lower MBC/MBEC value (1.25) for WT strain as compared with MBC/MBDC (2.25), it still falls into the category of biofilm-eradicating agents, unlike the conventional antibiotics we tested (Garrison and Huigens, 2017). ...
... We showed here that DMNP used in combination with either streptomycin or rifampicin significantly enhanced their killing effect on M. smegmatis cells by interfering with persister cell formation ( Figure S3). Furthermore, DMNP, unlike the conventional antibiotics, can be viewed as a biofilm-eradicating agent characterized by a favorable MBC/MBDC ratio (about 2-3) ( Figure 7C) or MBC/MBEC ratio of 1.25 (Table S3) (Garrison and Huigens, 2017). Therefore, antipersistent substances elaborated on the basis of DMNP could be used potentially in combination with conventional antibiotics to eradicate chronic infections, including tuberculosis. ...
Article
Bacterial persistence coupled with biofilm formation is directly associated with failure of antibiotic treatment of tuberculosis. We have now identified 4-(4,7-DiMethyl-1,2,3,4-tetrahydroNaphthalene-1-yl)Pentanoic acid (DMNP), a synthetic diterpene analogue, as a lead compound that was capable of suppressing persistence and eradicating biofilms in Mycobacterium smegmatis. By using two reciprocal experimental approaches – Δ rel Msm and Δ relZ gene knockout mutations versus rel Msm and relZ overexpression technique – we showed that both Rel Msm and RelZ (p)ppGpp synthetases are plausible candidates for serving as targets for DMNP. In vitro, DMNP inhibited (p)ppGpp-synthesizing activity of purified Rel Msm in a concentration-dependent manner. These findings, supplemented by molecular docking simulation, suggest that DMNP targets the structural sites shared by Rel Msm, RelZ, and presumably by a few others as yet unidentified (p)ppGpp producers, thereby inhibiting persister cell formation and eradicating biofilms. Therefore, DMNP may serve as a promising lead for development of antimycobacterial drugs.
... Phases of bacterial growth. Adapted fromGarrison & Huigens (2017). ...
Thesis
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This study aimed to consolidate a strategy to valorise immature tomato fruit (GT, cv. H1015) through controlled fermentation (use of starter cultures) in producing high-value food products to support circular economy-oriented innovation. The probiotic character of two pure LAB strains, Lactiplantibacillus plantarum (LAB97, isolated from GT) and Weissella paramesenteroides (C1090, INIAV collection), were tested using static in vitro gastrointestinal digestion model (sequential digestion and digestive enzymes). Both LAB strain counts reached ca. 6 log CFU/ml after the in vitro simulation, meeting the viability criterion for potential probiotic capacity. In the evaluation of GT-controlled fermentation, the two starters (per se) and the addition of NaCl (1.5%) were assessed (108 CFU/ml of inoculum, 100 rpm, 20 °C, 14 days). It was concluded that LAB 97 strain was superior to the C1090 strain or spontaneous fermentation because it increased process efficiency (fast acidification) and developed an ingredient with sensory acceptance and probiotic potential (> 7 log CFU/ml). The second approach aimed to evaluate the formulation of a sauce with sensory, nutritional, and probiotic potential based on the combination of fermented GT (LAB 97) with other valuable ingredients (avocado, parsley, and honey). The formula chosen included fermented GT (65%) and a 4:2:1 mixture of these ingredients. Different technological strategies (thermal treatment and non-treatment) were tested to prevent microbial contamination by the additional ingredients and promote the shelf life of the sauce storage. The sauce’s shelf stability samples were evaluated during storage (5 °C, 21 days) concerning several quality attributes (microbial counts, pH, soluble solids content, CIELab, total phenolic content, and antioxidant activity and panel sensory analysis). The viability of a sauce prototype with sensory quality and valuable antioxidant composition, meeting the microbiological criteria for this type of product, could be concluded. However, decontamination treatments do not improve sauce stability compared to raw ingredients.
Thesis
Lactic acid bacteria (LAB) are widely used to produce a variety of fermented and functional foods. On an industrial scale, LAB are produced and stabilized by freezing and freeze-drying. Depending on the strain, these cryopreservation processes lead to more or less significant degradation of their functional properties. Consequently, some LAB strains, such as L. bulgaricus CFL1, remain unexploited on an industrial scale. Different strategies exist to limit this degradation: the first one consists of modifying the fermentation conditions at the expense of the quantity produced, and the second strategy consists of adding a sugar solution.In this context, L. bulgaricus CFL1, a strain sensitive to cryopreservation, was used as a model lactic acid bacterium. This thesis aimed at identifying the fermentation condition that improved the resistance of L. bulgaricus CFL1 by implementing two strategies and analyzing their effects on the cell membrane, identified as the primary site of damage. The first strategy was to optimize the fermentation conditions to achieve a compromise between fair biomass production and high resistance to freezing and freeze-drying, using multi-objective optimization. There is no unique fermentation condition that satisfied both the biomass and functional properties requirements. Fermentation conditions had to be adapted to the chosen stabilization process.Each of these fermentation conditions impacted the lipid membrane's properties, which were then related to the cryopreservation properties of the bacteria. Here, the membrane characteristics depended on the stabilization process. For enhanced freezing resistance, fermentation conditions that generated more unsaturated fatty acids and a more fluid membrane were beneficial. For freeze-drying, conditions that favored cyclic fatty acid production contributed significantly to maintaining functional properties.The second strategy involved the selection of a protective sugar solution among glucose molecules of different degrees of polymerization. The results showed that the optimal choice of sugar depended on the stabilization processes.Therefore, this thesis project opens up new approaches for producing LAB: 1) outside the optimal production conditions of a bacterium, some conditions are slightly less productive but very beneficial for the maintenance of their functional properties. Thus, the choice of the stabilization process should be made upstream to target the appropriate fermentation conditions and protective sugar solution. 2) The formation of unsaturated or cyclic fatty acids in the membrane lipids will help preserve the bacterium from the damage occurring during freezing and freeze-drying.
Thesis
Bien que la thérapie photodynamique a été découverte il y a plus d’un siècle par sa capacité à inactiver les microorganismes, elle a été développée principalement comme traitement thérapeutique anticancéreux. Récemment, avec le nombre croissant d'infections causées par des bactéries multirésistantes, la chimiothérapie photodynamique antimicrobienne (PACT) est considérée comme une approche antimicrobienne alternative prometteuse. La PACT est une stratégie thérapeutique non invasive, et a une action rapide. De plus, la PACT ne semble pas induire la mise en place de mécanismes de résistance par les bactéries, ce qui en fait une alternative attrayante, par exemple, aux traitements conventionnels des infections des plaies. Les photosensibilisateurs les plus utilisés dans le PACT sont les porphyrines et leurs dérivés. Cependant, ces composés souffrent d’une faible solubilité dans l'eau, d’auto-quenching et d’un manque de sélectivité contre les cellules bactériennes. Afin de pallier certains problèmes liés à l’utilisation des porphyrines en tant que photosensibilisateurs dans la PACT, nous nous sommes intéressés, au cours de ce travail, à deux stratégies pour l’optimisation de cette thérapie. La première consiste à coupler une porphyrine à deux dérivés de maltodextrines (maltohexaose ou maltotriose) utilisés récemment comme agent de ciblage bactérien pour l'imagerie médicale , afin d’améliorer la sélectivité de la porphyrine vis-à-vis les cellules bactériennes. L’évaluation biologique de ces conjugués a montré que l’association des porphyrines avec les maltooligosacharide augmente leur efficacité antibactérien Staphylococcus aureus et Staphylococcus epidermidis. La deuxième stratégie vise à incorporer des porphyrines dans des hydrogels à base de xylane. Pour cela, trois voies ont été explorées : dans une première approche, l’hydrogel a été synthétisé d’abord par réticulation de xylane avant de le chargé par une porphyrine cationique. Dans la deuxième, les porphyrines sont fixées d’abord par liaisons covalentes sur le xylane, puis les hydrogels ont été obtenus à partir de ces xylanes fonctionnalisés par un agent de réticulation. Dans la troisième méthode, l’hydrogel est obtenu directement par une réticulation directe du xylane par les porphyrines. Tous les hydrogels obtenus ont montré une bonne intégrité mécanique et un taux de gonflement élevé et une forte activité photoantibactérienne vis-à-vis des bactéries Gram positif et Gram négatif.
Thesis
Après l’âge d’or de la découverte des antibiotiques, les infections bactériennes représentent toujours un challenge considérable pour la santé publique mondiale. Le mode de croissance en biofilms est le principal responsable des infections chroniques pour lesquelles les thérapies antibiotiques sont en échec clinique, pointant la nécessité d’élaborer de nouvelles stratégies thérapeutiques. L’objectif de cette thèse a porté sur le développement d’une thérapie combinatoire à base d’agents anti-biofilms et de particules biodégradables d’acide poly-lactique pour la délivrance d’antibiotiques au cœur des biofilms bactériens. Une stratégie de formulation d’antibiotiques a été développée, aboutissant à des particules stables et fortement chargées en rifampicine. La charge de surface des particules a été inversée vers des valeurs positives par adsorption d’un peptide cationique, la poly-lysine. L’intérêt d’une telle formulation a été évalué in vitro sur des biofilms de Staphylococcus aureus. Capables d’interagir via des liaisons électrostatiques avec les biofilms et les bactéries, les particules cationiques sont retenues en plus grande quantité dans les biofilms que les particules anioniques qui peuvent être éliminées par lavage. Les particules permettant une délivrance progressive de l’antibiotique, l’inversion de leur charge de surface qui renforce ces interactions permet de réduire les quantités d’antibiotique nécessaires pour maintenir une efficacité anti-biofilm. La combinaison de ce traitement avec la DNase, une enzyme capable de dégrader la matrice de biofilms, permet de potentialiser la dégradation des biofilms sans toutefois augmenter l’activité bactéricide de l’antibiotique. Des évaluations in vivo de l’efficacité de cette stratégie thérapeutique permettraient de confirmer son intérêt pour le traitement des biofilms bactériens.
Book
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https://singipedia.singidunum.ac.rs/izdanje/43755-osnove-tehnologije-zivotnih-namirnica
Article
Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of N-aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (e.g., HP 29, against methicillin-resistant Staphylococcus aureus: MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs 3, 28, and 29 induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against Mycobacterium tuberculosis (HP 34, MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP 29 demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.
Article
The recalcitrance exhibited by microbial biofilms to conventional disinfectants has motivated the development of new chemical strategies to control and eradicate biofilms. The activities of several small phenolic compounds and their trichloromethylsulfenyl ester derivatives were evaluated against planktonic cells and mature biofilms of Staphylococcus epidermidis and Pseudomonas aeruginosa. Some of the phenolic parent compounds are well-studied constituents of plant essential oils, for example, eugenol, menthol, carvacrol, and thymol. The potency of sulfenate ester derivatives was markedly and consistently increased toward both planktonic cells and biofilms. The mean fold difference between the parent and derivative minimum inhibitory concentration against planktonic cells was 44 for S. epidermidis and 16 for P. aeruginosa. The mean fold difference between the parent and derivative biofilm eradication concentration for 22 tested compounds against both S. epidermidis and P. aeruginosa was 3. This work demonstrates the possibilities of a new class of biofilm-targeting disinfectants deploying a sulfenate ester functional group to increase the antimicrobial potency toward microorganisms in biofilms.
Article
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The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response, Page 1 of 2 Abstract The staphylococci comprise a diverse genus of Gram-positive, nonmotile commensal organisms that inhabit the skin and mucous membranes of humans and other mammals. In general, staphylococci are benign members of the natural flora, but many species have the capacity to be opportunistic pathogens, mainly infecting individuals who have medical device implants or are otherwise immunocompromised. Staphylococcus aureus and Staphylococcus epidermidis are major sources of hospital-acquired infections and are the most common causes of surgical site infections and medical device-associated bloodstream infections. The ability of staphylococci to form biofilms in vivo makes them highly resistant to chemotherapeutics and leads to chronic diseases. These biofilm infections include osteomyelitis, endocarditis, medical device infections, and persistence in the cystic fibrosis lung. Here, we provide a comprehensive analysis of our current understanding of staphylococcal biofilm formation, with an emphasis on adhesins and regulation, while also addressing how staphylococcal biofilms interact with the immune system. On the whole, this review will provide a thorough picture of biofilm formation of the staphylococcus genus and how this mode of growth impacts the host.
Article
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Persistent bacteria, including persister cells within surface-attached biofilms and slow-growing pathogens lead to chronic infections that are tolerant to antibiotics. Here, we describe the structure-activity relationships of a series of halogenated phenazines (HP) inspired by 2-bromo-1-hydroxyphenazine 1. Using multiple synthetic pathways, we probed diverse substitutions of the HP scaffold in the 2-, 4-, 7- and 8-positions providing critical information regarding their antibacterial and bacterial eradication profiles. Halogenated phenazine 14 proved to be the most potent biofilm-eradicating agent (≥99.9% persister cell killing) against MRSA (MBEC < 10 µM), MRSE (MBEC = 2.35 µM) and VRE (MBEC = 0.20 µM) biofilms while 11 and 12 demonstrated excellent antibacterial activity against M. tuberculosis (MIC = 3.13 µM). Unlike antimicrobial peptide mimics that eradicate biofilms through the general lysing of membranes, HPs do not lyse red blood cells. HPs are promising agents that effectively target persistent bacteria while demonstrating negligible toxicity against mammalian cells.
Article
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Small molecules capable of eradicating non-replicating bacterial biofilms are of great importance to human health as conventional antibiotics are ineffective against these surface-attached bacterial communities. Here, we report the discovery of several halogenated quinolines (HQs) identified through a reductive amination reaction that demonstrated potent eradication of MRSA (; MBEC = 125 μM), MRSE (; MBEC = 3.0 μM) and VRE (, and ; MBEC = 1.0 μM) biofilms. HQs were evaluated using the Calgary Biofilm Device (CBD) and demonstrated near equipotent killing activities against planktonic and biofilm cells based on MBC and MBEC values. When tested against red blood cells, these HQ analogues demonstrated low haemolytic activity (3 to 21% at 200 μM) thus we conclude that these HQ analogues do not operate primarily through the destruction of bacterial membranes, typical of other biofilm-eradicating agents (i.e., antimicrobial peptides). HQ antibacterial agents are potent biofilm-eradicating compounds and could lead to useful treatments for biofilm-associated bacterial infections.
Article
The global burden of antibiotic resistance is tremendous and, without new anti-infective strategies, will continue to increase in the coming decades. Despite the growing need for new antibiotics, few pharmaceutical companies today retain active antibacterial drug discovery programmes. One reason is that it is scientifically challenging to discover new antibiotics that are active against the antibiotic-resistant bacteria of current clinical concern. However, the main hurdle is diminishing economic incentives. Increased global calls to minimize the overuse of antibiotics, the cost of meeting regulatory requirements and the low prices of currently marketed antibiotics are strong deterrents to antibacterial drug development programmes. New economic models that create incentives for the discovery of new antibiotics and yet reconcile these incentives with responsible antibiotic use are long overdue. DRIVE-AB is a €9.4 million public–private consortium, funded by the EU Innovative Medicines Initiative, that aims to define a standard for the responsible use of antibiotics and to develop, test and recommend new economic models to incentivize investment in producing new anti-infective agents.
Article
Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm-associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2-position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2-position. We identified diverse analogues that are alkylated and aminated at the 2-position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μm) and biofilm eradication (MBEC 1.0-93.8 μm) activities against drug-resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μm. In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram-positive pathogens that could lead to useful treatments against persistent bacterial infections.
Article
The 2-position of the halogenated quinoline (HQ) scaffold can be dramatically tuned through diverse and practical synthetic pathways, including reductive amination and alkylation reactions. New HQs discovered during these investigations potently eradicate methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE) and vancomycin-resistant Enterococcus faecium (VRE) biofilms, which display high levels of tolerance towards conventional antibiotic therapies. The background image is reproduced with permission and copyright© of the British Editorial Society of Bone and Joint Surgery (Bone Joint J. 2013, 95B, 678–682; Figure 2 b). More information can be found in the Full Paper by R. W. Huigens III et al. (DOI: 10.1002/chem.201600926).
Article
The in vitro activity of dalbavancin was tested against biofilms of 171 staphylococci associated with prosthetic joint infection. Dalbavancin minimum biofilm bactericidal concentration (MBBC) values were: MBBC50 for Staphylococcus aureus and Staphylococcus epidermidis, 1 μg/mL; MBBC90 for S. aureus, 2 μg/mL; MBBC90 for S. epidermidis, 4 μg/mL.
Article
Chemical modification of synthetic or natural product antibiotic scaffolds to expand potency and spectrum and to bypass mechanisms of resistance has dominated antibiotic drug discovery and proven immensely successful. However, the inexorable evolution of drug resistance coupled with a drought in innovation in antibiotic discovery contribute to a dearth of new drugs entering to market. Better understanding of the physicochemical properties of antibiotic chemical space is required to inform new antibiotic discovery. Innovations such as the development of antibiotic adjuvants to preserve efficacy of existing drugs together with expanding antibiotic chemical diversity through synthetic biology or new techniques to mine antibiotic producing organisms, are required to bridge the growing gap between the need for new drugs and their discovery.
Article
Venous access catheters used in clinics are prone to biofilm contamination, contributing to chronic and nosocomial infections. Although several animal models for studying device-associated biofilms were previously described, only a few detailed protocols are currently available. Here we provide a protocol using totally implantable venous access ports (TIVAPs) implanted in rats. This model recapitulates all phenomena observed in the clinic, and it allows bacterial biofilm development and physiology to be studied. After TIVAP implantation and inoculation with luminescent pathogens, in vivo biofilm formation can be monitored in situ, and biofilm biomass can be recovered from contaminated TIVAP and organs. We used this protocol to study host responses to biofilm infection, to evaluate preventive and curative antibiofilm strategies and to study fundamental biofilm properties. For this procedure, one should expect ∼3 h of hands-on time, including the implantation in one rat followed by in situ luminescence monitoring and bacterial load estimation.
Article
Conventional antibiotics are ineffective against non-replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1, that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC=0.2-12.5 μM), as well as the effective killing of MRSA persister cells in non-biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non-biofilm persisters alongside 14. HP 13 displayed potent antibacterial activity against slow-growing M. tuberculosis (MIC=3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non-toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.