Download Quaternary ammonium surfactant structure determines selective

J Antimicrob Chemother 2016; 71: 641 – 654
doi:10.1093/jac/dkv405 Advance Access publication 17 December 2015
Quaternary ammonium surfactant structure determines selective
toxicity towards bacteria: mechanisms of action and clinical
implications in antibacterial prophylaxis
Ângela S. Inácio1, Neuza S. Domingues2, Alexandra Nunes3, Patrı́cia T. Martins4, Maria J. Moreno4, Luı́s M. Estronca1,
Rui Fernandes5, António J. M. Moreno6, Maria J. Borrego3, João P. Gomes3, Winchil L. C. Vaz2 and Otı́lia V. Vieira2*
1
CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2CEDOC, NOVA Medical School | Faculdade
de Ciências Médicas, Universidade NOVA de Lisboa, 1169-056 Lisboa, Portugal; 3Department of Infectious Diseases, National Institute of
Health, Lisbon, Portugal; 4Centro de Quı́mica de Coimbra and Departamento de Quı́mica, Universidade de Coimbra, 3004-535 Coimbra,
Portugal; 5IBMC/HEMS – Instituto de Biologia Molecular e Celular/Histology and Electron Microscopy Service, Universidade do Porto,
Porto, Portugal; 6Department of Life Sciences, University of Coimbra, Coimbra, Portugal
*Corresponding author. Tel: +00351 218 803 100, ext. 26021; E-mail: [email protected]
Received 22 July 2015; returned 6 October 2015; revised 2 November 2015; accepted 2 November 2015
Objectives: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them
attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically
transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the
mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects
antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections.
Methods: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify
selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism
were evaluated to establish the mechanisms involved in selective toxicity of QAS.
Results: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium
bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and
.20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified:
impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and
electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher
concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic
cell death.
Conclusions: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large
therapeutic window to be good microbicide candidates.
Introduction
Quaternary ammonium surfactants (QAS) are amphiphilic molecules with a positively charged quaternary ammonium polar head
group having one or two apolar chains, usually n-alkyl, attached
to it. QAS are a sub-family of the so-called quaternary ammonium
compounds (QAC) with which they share some, but not all,
physico-chemical properties. QAS microbicidal activity has been
known since the mid-1930s1 and their use as disinfectants and
antiseptics for both general hygiene and clinical purposes has a
long history.2,3 The well-known broad-spectrum antimicrobial
activity of QAS combined with their low price, chemical stability
and non-demanding storage requirements make these compounds very attractive for use as general purpose disinfectants
and as antiseptics in topical prophylaxis of sexually transmitted
infections (STIs) and intrapartum transmission of urogenital infections from mother to neonate when adequate measures were not
taken prior to term. In the past two decades there have been a
great number of reports concerning the bactericidal activity of
surfactants against two of the major sexually transmitted pathogens, Chlamydia trachomatis and Neisseria gonorrhoeae.4 – 6
However, attempts to use surfactants as antiseptics in prophylaxis against STIs have led to spectacular failures in two sets of
clinical trials in the 1990s7,8 and early 2000s9,10 and drove most
academic and pharmaceutical interest away from exploring
them as prophylactic antiseptic microbicides. In the present
work, we argue that these studies were ill-devised and failed to
# The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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Inácio et al.
take into account the complexity of surfactant interactions with
biological membranes.
Given their amphiphilic nature, QAS partition favourably into
cell membranes, altering their physical properties and affecting
their function,11,12 which can result in cell death. A surfactant’s
concentration in cell membranes is, therefore, an important
determinant of its toxic effects. Surfactants are driven into biological membranes and all similar amphiphilic aggregates primarily by the hydrophobic effect. At concentrations above their molar
solubility in the membrane, non-ideal miscibility of surfactant and
phospholipids may lead to increased membrane porosity while
shape-factor mismatches, even under conditions of ideal miscibility, may affect membrane curvature- and/or area-elastic energies
and result in changes in membrane thickness.13 The resulting
‘hydrophobic mismatch’ of integrally associated membrane proteins14,15 affects membrane-related cellular biochemistry.11,16
When surfactant concentrations in the aqueous phase exceed
the critical micelle concentration (CMC), surfactant micelles coexist with surfactant-containing membranes and a dynamic equilibrium of membrane and micellar constituents results in eventual
membrane dissolution. None the less, although the microbicidal
activity of QAS has been recognized for many years, complete
details of the mechanisms behind it, beyond the fact that they
neutralize membrane charge17 and, at higher concentrations, dissolve bacterial membranes,18,19 are lacking. In this study, we
attempt to fill this gap to identify possible selective and potent
bactericidal agents.
We have demonstrated that, while incapable of inhibiting viral
infection at sub-lethal concentrations, monoalkyl QAS may function as bactericides at concentrations that are not harmful to
polarized mammalian epithelial cells, a property not shared by
other surfactant families.20 When used in high concentrations,
to disrupt bacterial membranes, surfactants show no selectivity,
also destroying the membrane of mammalian cells.21 However,
different sensitivities to the harmful effects of QAS at concentrations below the CMC may arise from distinct chemical composition,
physical properties and physiological functions of pathogens and
host cell membranes, as well as the total amount of membrane
per cell.20,22 Therefore, understanding QAS mechanisms of action
at concentrations below the CMC, in both eukaryotic and prokaryotic cells, is crucial for the design and development of more
effective and safer molecules.
In the present study, we show that the mechanisms underlying
QAS antimicrobial activity are quite distinct from those responsible for their toxic effects on epithelial cells and are dependent
on their molecular structure. We also show that some QAS have
therapeutic indices that encourage their development for use as
topical microbicides for diseases of bacterial aetiology.
Materials and methods
Reagents
QAS of the highest commercially available purity, decyltrimethylammonium bromide (C10TAB), dodecyltrimethylammonium bromide (C12TAB),
tetradecyltrimethylammonium bromide (C14TAB), hexadecyltrimethylammonium bromide (C16TAB), dodecyl-N-benzyl-N,N-dimethylammonium
bromide (C12BZK) and n-dodecylpyridinium bromide (C12PB), were
purchased from Sigma-Aldrich (St Louis, MO, USA) and used as received.
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) was obtained
from Avanti Polar Lipids (Alabaster, AL, USA). All mammalian cell culture
642
reagents were purchased from Gibcow Life Technologies S.A. (Paisley,
Scotland, UK), the Luria broth base (Miller’s LB broth base) was supplied by
InvitrogenTM Life Technologies S.A. (Carlsbad, CA, USA), agar-agar was
obtained from Merck (Darmstadt, Germany) and chocolate agar PolyViteX
plates from bioMérieux (Montreal, Quebec). For the N. gonorrhoeae assays
the Fastidious Broth (FB) medium used was prepared essentially as described previously by Cartwright et al.23 Propidium iodide, rhodamine 123
(Rh123), Hoechst 33342, FMw 4-64 dye [N-(3-triethylammoniumpropyl)4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide], Click-iTw
5-ethynyl-2′ -deoxyuridine (EdU) Alexa Fluorw 488 Imaging Kit and Live/
Deadw BacLightTM Bacterial Viability Kit were from Molecular Probesw
Invitrogen Corporation (Paisley, Scotland, UK). All other chemicals used
were from Sigma-Aldrich.
Determination of QAS partition coefficient (Kp) into
POPC membranes
The Kp of CnTAB (n¼10, 12 and 14), C12BZK and C12PB, between aqueous
HEPES buffer (0.01 M HEPES/0.15 M NaCl/0.001 M EDTA, pH 7.4) and
100 nm diameter unilamellar POPC liposomes, was measured by isothermal titration calorimetry using VP-isothermal titration calorimetry equipment from MicroCal (Northampton, MA, USA) at 378C with a minimum
of three independent titrations for each QAS (typically more than five).
The experimental protocol followed for the preparation of the liposomes,
titration experiments and data analysis has been described previously.24
The concentration of CnTAB was always well below their CMC (typically
20 mM for n¼10 and 12 and 5 mM for n¼14) leading to local concentrations in the lipid bilayer below 2%. At these concentrations and high ionic
strength in aqueous media, surface electrostatic effects resulting from the
charge of the QAS inserted into the lipid bilayers may be neglected and the
Kp were obtained considering simple partition, without correction for electrostatic effects.24 In the absence of well-defined values for the rate of
translocation, it was assumed that all lipid was available for interaction
with the QAS in the analysis of the results obtained from the isothermal
titration calorimetry experiments.
Antimicrobial susceptibility testing
Escherichia coli was isolated from human necropsies and identified by API
20E system (bioMérieux) as 5144573 biotype. The strain is resistant to
ampicillin, trimethoprim, ofloxacin, norfloxacin and ciprofloxacin, and susceptible to amoxicillin/clavulanic acid, cefuroxime, nitrofurantoin, fosfomycin, gentamicin, cefotaxime, ceftazidime, ceftriaxone, amikacin,
aztreonam, netilmicin, imipenem and piperacillin/tazobactam. Before
each experiment, frozen stocks were subcultured at least once to check
strain viability. Briefly, a frozen stock culture was inoculated in 10 mL of
LB, grown overnight in an orbital shaker (200 rpm) at 378C, and then
diluted 1:100 (v/v) in 10 mL of fresh LB. When the late logarithmic growth
phase was reached, an E. coli inoculum was prepared by direct broth suspension. The MIC of each QAS assayed was determined by the broth microdilution method,25,26 using an inoculum of 1.5×10 8 cfu/mL (turbidity
equivalent to that of a 0.5 McFarland standard) or 7.5×106 cfu/mL (1:20
dilution). Two-fold serial dilutions of concentrated stock QAS solutions
were prepared in LB medium in 96-well multiwell plates. A control without
QAS was also prepared. All cultures were incubated for 18 h in an orbital
shaker (200 rpm) at 378C. Purity check and colony counts of the inoculum
suspensions were also evaluated to ensure that the final density closely
approximated the intended number. The MIC was determined as the lowest QAS concentration at which no visible growth was observed.
Determination of QAS bactericidal activity
E. coli cell suspensions (1.5×108 cfu/mL) prepared from a late logarithmic
growth phase were exposed for 10, 20, 60 and 120 min to different
JAC
Quaternary ammonium surfactants as microbicides
concentrations of QAS in LB, at 378C in an orbital shaker (200 rpm). At the
end of incubation an aliquot of each sample was serially diluted in LB to
neutralize surfactant activity and spread in LB-agar plates using the
drop method as described by Miles et al.27 The plates were incubated
at 378C for 8 – 10 h and the number of visible colonies per plate was
counted and expressed as the percentage of mock-treated bacteria,
to determine the percentage of survival of culturable bacteria.28,29
Bacterial cell suspensions (turbidity equivalent to that of a 2 McFarland
standard) of N. gonorrhoeae PT07-15 and N. gonorrhoeae PT10-12 (both
from the Portuguese NIH National collection) were prepared in FB from a
48 h plate. The bacterial cultures were then diluted 1:100 (v/v) in 1 mL of
fresh FB and grown for 60 min in the absence or presence of different
C12PB concentrations, at 378C and 5% CO2. At the end of incubation,
samples were serially diluted in FB to neutralize surfactant activity and
spread in chocolate PVX agar plates. The plates were incubated at 378C
and 5% CO2 for 48 h, and the number of visible colonies on each plate
was counted and expressed as a percentage of control cultures.
prepared using a diamond knife (Diatome, Hatfield, PA, USA) and were
recovered to 200 mesh Formvar Ni-grids. Staining of sections using
2 wt% uranyl acetate and saturated lead citrate solution, for 7 min
each, was performed before observation. Visualization took place at
80 kV in a JEOL JEM 1400 microscope (Japan).
EdU labelling of replicating DNA in E. coli
To label replicating bacterial cells, 30 mg/mL EdU was added to each
sample 15 min before the end of 1 h of incubation with QAS.33 Cells
were then fixed overnight with 4% paraformaldehyde at 48C and permeabilized with 0.1% Triton X-100, followed by EdU staining according to the
manufacturer’s instructions. The percentage of replicating cells was determined by flow cytometry. For microscopy analysis, after EdU staining cells
were also labelled with 5 mg/mL FM4-64 and 2 mg/mL Hoechst 33342 for
30 min at room temperature, to stain cell membranes and nucleoid,
respectively, and immobilized on 1% agarose for immediate visualization
in a Confocal Microscope LSM 510.
Evaluation of E. coli membrane integrity
Bacterial membrane integrity was assessed using the Live/Dead w
BacLightTM Bacterial Viability Kit30 after 1 h of incubation with QAS, as
described above. At the end of incubation, bacteria were centrifuged at
6000 g for 5 min and resuspended in PBS. SYTO 9 and propidium iodide
were mixed in a 1:1 proportion and 3 mL was added to 1 mL of each sample, followed by 15 min of incubation in the dark at room temperature.
Samples were analysed by flow cytometry in a Becton Dickinson
FacsCalibur (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions. 30 000 events were collected per sample. Data
were analysed using CELLQuest (BD Biosciences) or Flowing Software
Version 2.5.1, a public domain program developed by Perttu Terho (Turku
Centre for Biotechnology). The percentage of permeabilized propidium
iodide+ cells was determined and bacterial viability is expressed as the
percentage of untreated control cultures.
E. coli cell length measurements
After 1 h of incubation with QAS, E. coli cell suspensions were washed
and resuspended in PBS. For microscopy analysis, bacterial cells were immobilized on 1% agarose for immediate visualization in a Carl Zeiss Laser
Scanning Confocal Microscope LSM 510 (Carl Zeiss Inc., Oberkochen,
Germany). Differential interference contrast (DIC) images were acquired
with a Plan-Apochromat ×63 oil immersion objective (numerical
aperture¼1.40) using the Carl Zeiss Laser Scanning System LSM 510 software. DIC images were used for bacterial cell length determination with
the public domain program Coli-Inspector running under plugin ObjectJ
developed by Norbert Vischer (University of Amsterdam, http://www.simon.
bio.uva.nl/objectj), which runs in combination with ImageJ (http://www.
imagej.nih.gov/ij/).31
Transmission electron microscopy
For electron microscopy, E. coli was grown and treated with different C12PB
concentrations for 1 h. At the end of incubation, bacterial cells were
washed with 0.1 M sodium cacodylate (pH 7.2), pre-fixed overnight with
1.25% glutaraldehyde plus 4% paraformaldehyde at 48C, washed
again with sodium cacodylate and fixed with 2% osmium tetroxide in
veronal-acetate buffer (pH 6.2) for 2 h at room temperature, followed by
washing in distilled water and post-fixing with 1% uranyl acetate for
30 min at room temperature.32 Next, samples were dehydrated in a
graded series of ethanol and propylene oxide as follows: 70% ethanol
for 10 min, 90% ethanol for 10 min and 100% ethanol for 30 min repeated
four times followed by 10 min immersion in propylene oxide. Lastly, samples were embedded in Epon resin. Fifty nanometre thick sections were
E. coli DNA content and cell cycle analysis
DNA content per cell and cell cycle were determined by flow cytometry as
described previously,33 with some modifications. After 1 h of incubation
with C12PB, bacterial cells were fixed overnight in 70% ethanol at 48C, centrifuged to remove the ethanol, washed twice with 50 mM sodium citrate
pH 7.5 and resuspended in the same buffer. Bacteria where then treated
with RNase A at a final concentration of 250 mg/mL for 1 h at 378C. DNA
was stained with 2.5 mM SYTO 9 in PBS for 1 h at room temperature. To
evaluate the effect of C12PB on bacterial cell size, which is proportional
to the forward light-scattering signal, an aliquot of each sample was collected before fixation and analysed immediately. To examine the bacterial
cell cycle, after C12PB incubation, cultures were grown for a further 3 h at
378C in run-out conditions.33,34 At the end of C12PB incubation, cells were
washed and re-suspended in LB medium supplemented with 300 mg/L
rifampicin and 30 mg/L cefalexin.33 After that, samples were processed
and stained as described above. Cells grown in M9 medium supplemented
with 0.2% of glucose, which cycle between 1N and 2N DNA content, were
used to infer ‘N’ positions in the fluorescence intensity x-axis (linear scale).
Assessment of bacterial membrane potential
Analysis of E. coli membrane potential was performed using the membrane potential-sensitive probe Rh123, as described elsewhere.35 Briefly,
after 1 h of incubation with QAS, cells were washed and re-suspended in
PBS with 10 mg/L Rh123 and incubated at 378C for 30 min, after which
cells were washed again and re-suspended in PBS for flow cytometry
analysis. Rh123 uptake and accumulation in the bacterial cytoplasm, in
response to the electrical potential across the plasma membrane, induces
fluorescence quenching so that an increase in fluorescence corresponds
to membrane depolarization. As a positive control, membrane depolarization was induced by treating E. coli with 10 mM carbonyl cyanide
4-(trifluoromethoxy)phenylhydrazone (FCCP) or 5 mM potassium cyanide
for 10 min at 378C before cell labelling.
Quantification of intracellular adenine nucleotide levels
Intracellular ATP, ADP and AMP levels were determined by ion-pair reversephase HPLC. After 1 h of exposure to QAS, E. coli cultures were centrifuged
at 6000 g for 5 min and re-suspended in cold PBS. An aliquot of the sample
was taken for protein quantification by the bicinchoninic acid method
(Piercew BCA Protein Assay Kit). The remaining sample was further handled
for intracellular adenine nucleotide acid extraction (perchloric acid) as
described elsewhere.36 Reversed-phase HPLC separation was performed
at 208C according to Folley et al.,37 using a C-18 (15 cm×4.6 mm) 5 mm
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Inácio et al.
The effect of QAS on E. coli aerobic respiration was measured in inside-out
membrane vesicles prepared from protoplasts as described by Futai38
Briefly, E. coli was grown to an OD of 0.9– 1.0 at 450 nm and protoplasts
were prepared using lysozyme and EDTA as described elsewhere,39 collected by centrifugation and then incubated for 15 min with 10 mg/L
DNase and 10 mg/L RNase A (in 0.01 M Tris/10 mM MgCl 2, pH 7.4) at
378C. The mixture was then passed through a French press (2000 psi).
After centrifugation, vesicles were re-suspended in 0.01 M Tris-HCl and
10 mM MgCl2, pH 7.4. Oxygen consumption by E. coli inverted membrane
vesicles was measured polarographically with a Clark-type oxygen electrode fitted to a 1 mL water-jacketed closed chamber, at 378C in a buffer
containing 10 mM Tris/10 mM MgCl2, pH 7.4. The experiments were performed using 1 mg of protein. Vesicles were incubated with different concentrations of QAS for 5 min prior to energization with 1 mM NADH or with
7.5 mM succinate.
Cell culture and MTT assay
The human intestinal columnar epithelial cell line C2BBe1 (ATCCw
CRL-2102TM ), a clone of Caco-2 cells, was grown for 5 days in DMEM
with GlutaMAXTM , supplemented with 10% FBS, 100 U/mL penicillin,
100 mg/mL streptomycin and 10 mg/L human transferrin, at 378C in a
humidified atmosphere containing 5% CO2. After that, cells were confluent and fully polarized.40 Cells were then incubated with increasing QAS
concentrations for 20, 60 and 120 min. Stock solutions of surfactants
were prepared in OptiMEM cell culture medium, without serum and antibiotics, as multiples of the respective CMC. At the end of the incubation,
QAS-containing medium was collected and replaced by fresh complete
culture medium without phenol red. Cell viability was assessed 24 h
after exposure to QAS by the MTT assay.41 The samples were quantified
colorimetrically at 570 nm (background correction at 620 nm) on a
SpectraMax Plus384 microplate spectrophotometer (Molecular Devices
Inc.). The background absorbance (culture medium plus MTT without
cells) was subtracted from the absorbance of each sample and data are
shown as a percentage of the control.
Statistical analysis and curve fitting
Results are expressed as mean+SD, unless otherwise stated. Statistical
analysis was carried out in GraphPad PRISMw software version 5.0 and
performed as described in the figure legends.
Dose – response toxicity curves were fitted by a weighted sum of
processes, each of which could be independently described by the Hill
equation:42
% CV = DCVmax −
fi × DCVmax ×
[QAS]x
,
x
[QAS]x + IC(50)i
where: % CV is cell viability relative to control and is by definition 100% in
the untreated control; DCVmax is the difference between the % CV (usually
100%) at the lowest non-toxic QAS concentration and the % CV (usually
0%) in the presence of a maximally toxic QAS concentration; fi is the
fractional contribution of each toxic process; [QAS] is the concentration
of surfactant to which the organism was exposed; IC(50)i is the
644
Results and discussion
Comparative studies of QAS antimicrobial efficacy
must take into account the surfactant CMC
QAS concentration at the site(s) of action will be influenced by
both its lipophilicity and that of each cell compartment, as well
as by the duration of exposure, which relates to the kinetics of
insertion and desorption from membranes and translocation
across them.43 – 46 Hence, a compromise between the capacity
of surfactants to partition between aqueous and membrane
phases and translocate across the latter is required for the
transport of the QAS to their site(s) of action, to achieve maximal
antimicrobial activity.
In principle, the membrane/water Kp informs us how well a
given QAS will partition into membranes. Determining Kp, however, requires methods that are not accessible to most laboratories, but Kp are expected to be related to the CMC—a measure
much easier to assess47 —in a straightforward manner.48,49
Figure 1(a) shows how Kp and CMC for the QAS used in this work
are related. As already mentioned, for most surfactants, gross
membrane dissolution occurs at concentrations close to the
CMC while more subtle effects that could discriminate between
mammalian and bacterial cell membranes may occur at somewhat lower concentrations. Indeed, one important aspect previously overlooked in pre-clinical and clinical studies was that
surfactant concentrations in the aqueous phase close to or
above the CMC result in indiscriminate membrane disruption.
Consequently, all the surfactant-based microbicide candidates
that completed Phase III clinical trials [nonoxynol-9 and
SAVVYw (C31G) vaginal gel] failed to prevent HIV infection7 – 10
since the concentrations used caused disruption of the epithelial
barrier, providing a direct access of HIV to the lamina propia where
virus target cells are more abundant.50 Thus, when surfactants
are used in disinfection or as antiseptics, their antimicrobial
(a)
C12BZK
4.5
4.0
3.5
3.0
2.5
2.0
C14TAB
C12PB
C12TAB
C10TAB
1
2
3
Log 1/CMC (M)
4
(b)
CMC or MIC (µM)
Preparation of E. coli inverted membrane vesicles and
measurement of oxygen consumption
concentration of surfactant at which the % CV is 50% of control for
Process i if that process were the only toxic process; and x is the Hill coefficient, which describes the sigmoidicity of the curve. The inhibitory concentrations, IC10, IC50 and IC90, were calculated from the theoretical
curves for each dataset.
Log Kp
analytical column combined with a suitable C-18 (4.6 mm×12.5 mm)
5 mm guard column. Separation was performed at a flow rate of 1.5 mL/
min and ultraviolet detection at 254 and 280 nm.36 Stock solutions of ATP,
ADP and AMP prepared in water were used for calibration. Peak identity
was determined based on the retention time and spectrum. Data were
normalized to protein content.
100 000
10 000
1000
100
10
1
CMC
MIC
8 10 12 14 16 18
n-Alkyl chain length
(number of C atoms)
Figure 1. Micelle formation and QAS partitioning into membranes. (a)
Correlation between membrane/water Kp and CMC for the QAS used in
this work. The linear dependence of log Kp on log 1/CMC for the CnTAB
family (filled circles) is shown. A similar correlation is expected for the
other analogous QAS families (open square, C12PB; open triangle,
C12BZK). The CMC values for the QAS used have been previously reported
by Inácio et al. 22 (b) Dependence of CMC and MIC for E. coli on n-alkyl
chain length for the CnTAB family. CMC and MIC are plotted on a
logarithmic scale.
JAC
Quaternary ammonium surfactants as microbicides
efficacy should be reported relative to their CMC as a reference
concentration. In the present work, all QAS concentrations will
be referred to the respective CMC, the values for which we have
previously reported.22
Antimicrobial activity of QAS
The antimicrobial activity of several mono-n-alkyl-QAS against
an MDR strain of E. coli was first evaluated by determining the
MICs. Since the most common bacterial STIs are caused by
Gram-negative bacteria (e.g. Treponema pallidum, N. gonorrhoeae
and C. trachomatis),51 E. coli was chosen as a model organism as it
is a non-fastidious Gram-negative bacterium of rapid growth,
allowing for greater experimental flexibility. Two characteristics
of QAS structure were examined, i.e. (i) effect of the apolar
n-alkyl chain length, and (ii) effect of the polar head structure.
The n-alkyl chain length of the trimethylammonium bromide
(TAB) family of QAS was varied between 10 and 16 carbon
atoms and three analogous QAS families [TAB, benzalkonium
bromide (BZK) and pyridinium bromide (PB)] with the same (C12)
n-alkyl chain were compared. MIC results are summarized in
Table 1. Since one of the critical factors that may affect the MIC
is the inoculum size,25 two inocula with a 20-fold difference in
the number of microorganisms were used. No impact on the measured MIC was observed.
The results in Figure 1(b) show that, as expected, for the CnTAB
family there is a linear correlation between the logarithm of a surfactant CMC and the n-alkyl chain length.48,49 However, a similar
correlation is not observed for the MIC. C10TAB and C12TAB inhibit
bacterial cell growth at concentrations that are less than onetenth of their CMC, whereas the MICs obtained for C14TAB and
C16TAB are very close to or above the respective CMC. This suggests that the C14 and C16 homologues exert their antibacterial
activity by causing gross membrane disarrangement or even its
dissolution. Early electron microscopic studies showed that bactericidal concentrations of C16TAB induced cytolytic damage and
cell leakage in Staphylococcus aureus, Streptococcus faecalis and
E. coli.19 On the other hand the C10 and C12 homologues most
likely exert their antimicrobial effects through one or several
more subtle mechanisms, such as immiscibility-induced increase
in membrane porosity or alteration of membrane elastic properties, making them better candidates for discriminatory toxicity
against bacterial cells as compared with eukaryotic cells.
Table 1. Antimicrobial susceptibility of E. coli to QAS
QAS
C10TAB
C12TAB
C14TAB
C16TAB
C12BZK
C12PB
MIC (mM)
MIC/CMC
3000
300
150
80
60
50
0.075
0.086
0.517
3.077
0.035
0.013
To evaluate the effect of the hydrophobic chain length and of the polar
head structure on the antibacterial efficacy of QAS, MICs are expressed
relatively to the respective CMC. Two inocula with different initial size
(1.5×108 cfu/mL and 7.5×106 cfu/mL) were prepared and no difference
in the measured MICs was observed.
When MICs were normalized with respect to the corresponding CMC (Table 1) the relative antibacterial efficacy was
C 12 PB .C 12 BZK .C 12 TAB. C 10 TAB and C 12 TAB have similar
MIC/CMC ratios, but as the antibiotic concentration of C10TAB is
10-fold higher than that of C12TAB, the C12 homologue is a
more attractive candidate. The PB analogue was about 4 – 9
times more effective in inhibiting E. coli growth than the BZK
and TAB analogues, respectively. The greater efficacy of the PB
analogue can be understood in terms of two properties, both of
which are related to the larger polar head with greater charge
delocalization: the larger polar head increases the molecular
area at the membrane–water interface, increasing the molecular
cone angle13 and leading to larger effects on membrane curvature elasticity;11,12 and the delocalized charge facilitates transmembrane translocation.52
In what follows, we shall present results with C12PB and
relegate all similar results with C12 TAB and C 12BZK to the
Supplementary data.
QAS antimicrobial activity involves different toxicity
mechanisms with different concentration and exposure
time dependences
Although the MIC is widely accepted as a measure of the antimicrobial activity of a drug, it offers no mechanistic information
or indication of whether the antimicrobial agent is bactericidal
or bacteriostatic. Thus, bactericidal activity of QAS was evaluated
by post-exposure inoculation and enumeration of cfu. Figure 2(a)
shows the dose– response toxicity plots for C12PB towards E. coli.
The curves show a multiphasic dose-dependent acute toxicity,
strongly suggesting that there are distinct processes responsible
for QAS bactericidal activity. Three toxic processes, all separated
by a plateau at intermediate concentrations, can be identified:
Process 1, at high C12PB concentrations; Process 2, at intermediate
concentrations; and Process 3 at low concentrations. Data were
fitted by a weighted sum of these three processes, each of
which could be independently described by the Hill equation42
(Figure S1, available as Supplementary data at JAC Online). The
fractional contribution of each toxic process to the overall toxicity
changes as a function of exposure time (Figure 2b) and can be
seen as a measure of the kinetics of the respective process.
Accordingly, Process 1 would have the fastest characteristic
reaction time and Process 3 the slowest. These results support
the hypothesis that the three processes result from distinct QAS
mechanisms of action. Curve fitting the data allowed us to estimate the concentrations at which the QAS was bactericidal
to 90% (IC90), 50% (IC50) and 10% (IC10) of the exposed bacterial
population relative to the control (Table S1). For data on C12TAB
and C12BZK see Figure S2.
To confirm that the toxicity pattern seen for E. coli was replicable in bacterial STI pathogens, similar experiments were
conducted with two strains of N. gonorrhoeae (Figure 2c). These
microorganisms are considerably more susceptible to C 12PB
than E. coli, but the overall characteristics of the curves are similar.
Considering the exposure time was 60 min, and by analogy to the
toxicity curves for E. coli, it is likely that the two processes observed
for N. gonorrhoeae correspond to Processes 2 and 3 seen for E. coli.
Depending on the strain, we note that N. gonorrhoeae was 2 – 7
times more susceptible to C12PB than E. coli.
645
Inácio et al.
CMC/X
10 000 3000 1000 300 100 30 10
(a)
3
1
(b)
1.0
Fractional contribution
cfu (% of control)
120
100
80
60
40
10 min
20 min
60 min
20
Process 1
Process 2
Process 3
0.8
0.6
0.4
0.2
0.0
0
0.1
1
10
100
1000
0
10 000
20
40
Time (min)
C12PB (µM)
CMC/X
10 000 3000 1000 300 100 30 10
(c)
3
1
Cell viability (% of control)
cfu (% of control)
PT10–12
PT07–15
120
CMC/X
1000 300 100 30 10
(d)
140
100
80
60
40
20
0
60
3
80
1
120
100
80
60
40
20 min
60 min
120 min
20
0
0.1
1
10
100
1000
10 000
C12PB (µM)
1
10
100
1000
10 000
C12PB (µM)
(e)
QAS therapeutic index for the tested microorganisms
Bacteria
E. coli
N. gonorrhoeae PT07–15
N. gonorrhoeae PT10–12
QAS
IC50(C2BBe1)/IC50(bacteria) after exposure time of
C12TAB
C12BZK
C12PB
C12PB
C12PB
20 min
8.2
4.3
7.2
–
–
60 min
6.2
8.4
11.6
76.4
21.2
Figure 2. QAS bactericidal activity. (a) E. coli cell suspensions were incubated with C12PB for the indicated times. Samples were then serially diluted,
spread in LB-agar plates and the survival of culturable bacteria was determined by counting the number of cfu/plate. Data are expressed as
percentage of untreated control cultures and presented as mean+SD of at least four independent experiments. C12PB concentration is plotted
on a logarithmic scale. The CMC of C12PB is represented by the black dashed line. A weighted sum of three Hill equations was fitted to the data
(lines, theoretical Hill plots). (b) Fractional contribution of the toxic processes occurring at high (Process 1), intermediate (Process 2) and low
(Process 3) concentrations. (c) Bactericidal activity of C12PB against two strains of N. gonorrhoeae after 60 min of exposure. Data are expressed
as percentage of untreated control cultures and presented as mean+SD of three independent experiments. C12PB concentration is plotted on a
logarithmic scale. (d) Effect of C12PB on the viability of polarized columnar epithelial cells (C2BBe1 cell line) as assessed by the MTT assay 24 h
after cells had been exposed to different C12PB concentrations for the indicated times. Cell viability is expressed as percentage of the viability of
control cells. Data are presented as mean+SD of at least three independent experiments, each one done in triplicate. A Hill equation was fitted
to the data (lines, theoretical Hill plots). C12PB concentration is plotted on a logarithmic scale. (e) Therapeutic indexes calculated after 20 and
60 min of incubation with C12PB.
Therapeutic indices of QAS
The major concern in the development of clinically useful microbicides for prophylaxis in transmission of STIs and perinatal urogenital infections is their selective toxicity against pathogens.
Ideally, a good microbicide should ensure a sound antimicrobial
646
activity while also being minimally toxic to the host. In the context
of surfactant use for STIs prophylaxis, it has been shown that the
vaginal columnar epithelium is the primary site of damage.7,53
The therapeutic potential of QAS was, therefore, evaluated by
comparing the bactericidal activity against and the toxic effects
Quaternary ammonium surfactants as microbicides
towards mammalian polarized epithelial cells. The C2BBe1 columnar epithelial cell line was used for this purpose. Although
not of vaginal origin, this cell line is derived from human columnar
epithelia and can be grown to a completely confluent and polarized state, with relatively non-leaky tight junctions,40 closely
resembling the characteristics of the vaginal columnar epithelium. In addition, the literature supports the use of this polarized
epithelial cell line in bacterial STI studies.40,54 – 56
As for bacterial cells, all tested QAS showed concentrationdependent and exposure-time-dependent toxic effects on epithelial cell cultures. However, all toxicity curves could be described by
a single component Hill equation (Figure 2d). This difference in the
shape of the toxicity curves strongly suggests that the mechanisms mediating QAS-induced toxicity are different for
prokaryotic and eukaryotic cells. The toxicity ranking for C2BBe1
epithelial polarized cells was C12PB ≈ C12BZK .C12TAB, which is
in agreement with our previous results for other mammalian
cell lines.22 The IC10, IC50 and IC90 calculated for each timepoint
are shown in Table S2.
The efficacy of QAS antibacterial activity was evaluated by the
therapeutic index, calculated as the ratio of the IC50 value for
polarized epithelial cells (C2BBe1) to the IC50 value for bacteria
(E. coli and N. gonorrhoeae), both treated with the same surfactant for the same exposure time.57,58 In agreement with previous
results, a larger polar head with a more delocalized positive
charge greatly enhances QAS activity against bacteria compared
with mammalian polarized epithelial cells (Figure 2e). C12PB was
the only QAS with therapeutic indices superior to 10 after a 60 min
exposure.
Overall, bacteria were more susceptible to QAS toxic effects
than mammalian epithelial cells. This difference in QAS susceptibility may originate from differences in lipid composition as well as
differences in the electrical potentials at membranes of the target
pathogen and host cells. Bacterial membranes are rich in negatively charged lipids whereas mammalian cells are mostly composed of zwitterionic lipids.59,60 Furthermore, the membrane
potential across the plasma membrane in eukaryotic cells is
more positive than in prokaryotic cells.61 – 63 As a result, adsorption
of cationic surfactants will preferentially occur on bacterial membranes. The cholesterol content in polarized epithelial cells, which
is absent in the bacterial membrane, is also known to make insertion into and translocation across the membrane,44,46 as well as
its disruption,64 more difficult. Moreover, the overall size and total
membrane surface is higher in mammalian cells than in bacteria,
thus confronted with the same QAS-containing aqueous phase,
the concentration of surfactant at its site(s) of action will be
more diluted in the mammalian cells, which may contribute to
the lower toxicity seen for these cells.
QAS inhibit E. coli colony formation without compromising
cell membrane integrity
Antimicrobial activity of surfactants is generally attributed to their
capacity to disorganize and disrupt cell membrane structure.2,11
Thus, the damage caused by C12PB on E. coli cell membrane integrity was studied by performing a dual staining with SYTO 9, a
membrane permeant, green-fluorescent dye that stains the
nucleic acids of both healthy and dead bacteria, and propidium
iodide, a red-fluorescent nucleic acid probe impermeant to
undamaged cell membranes that competes with SYTO 9 for
JAC
binding sites, causing a reduction in SYTO 9 fluorescence and
increase in propidium iodide fluorescence.30 Three distinct cell
populations were evident after 1 h of incubation with C12PB:
intact cells, SYTO 9+/propidium iodide2; partially damaged
cells, SYTO 9+/propidium iodide+; and severely damaged cells,
SYTO 92/propidium iodide+ (Figure 3a). Membrane integrity is
compromised at concentrations ≥CMC/100 and the extent of
the damage becomes more severe with increasing C12PB concentrations. The bacterial cluster on the two-dimensional dot plot
progresses in a curve shape, from a predominantly green fluorescence (SYTO 9) to a predominantly red fluorescence (propidium
iodide) with intermediate stages. Although our results present
no conclusive evidence to ascribe any particular sequence of
events, we note that Berney et al.30 obtained similar results for
Gram-negative bacteria submitted to EDTA treatment, which permeabilizes the outer membrane of these bacteria, but not for
Gram-positive bacteria under the same conditions. The authors
attributed their results to the existence of intermediate bacterial
viability states related to the degree of damage inflicted specifically at the bacterial outer membrane.
To visualize better the effects of QAS concentration on cell
membrane integrity, the percentage of propidium iodide+ cells
relative to untreated controls was determined for a wide range
of C12PB concentrations (Figure 3b). The percentage of propidium
iodide+ cells in control cultures was 1.0+0.9% (≈100% viable
cells). The dose – response toxicity curves obtained using propidium iodide staining were plotted together with the corresponding toxicity curves of viable and culturable cell counts to provide
further information concerning the relation between cell membrane damage and cell viability (Figure 3b). The propidium iodide+
QAS dose dependence revealed a biphasic sigmoid profile with
an overall toxicity described by the weighted sum of two Hill
equations, suggesting two distinct processes (Figure S1f). The
IC50 calculated for Processes 1 and 2 by either culture-dependent
or culture-independent methods are similar (Figure S1g), indicating that these two toxic processes result in a decrease in E. coli cfu
as a consequence of cell membrane damage. Notably, Process 3,
seen in viable cell counts, does not appear in the toxicity curves
obtained by propidium iodide staining. Process 3, therefore, does
not involve changes in membrane permeability. Detection of
viable bacteria performed by the classic colony count method is
limited to culturable bacteria, meaning that if QAS somehow
impaired cell growth or induced a quiescent state, commonly
referred to as ‘viable but non-culturable’, bacteria can fail to
grow on agar plates without necessarily implying that they are
metabolically inactive and/or dead. 65,66 Therefore, our results
suggest that the decrease in cell viability observed at lower QAS
concentrations actually corresponds to an impaired cell division,
possibly leading to cell cycle arrest and resulting in a diminished
capacity of bacteria to form colonies. In fact, at C12PB concentrations as low as CMC/300, there was no detectable propidium iodide staining despite the approximately 40% reduction in cfu. For
data on C12TAB and C12BZK see Figures S1 and S2.
To understand better the nature of Process 3, cells were treated
for 1 h with C12PB concentrations required to inhibit bacterial
growth by a maximum of 40% – 45%, and imaged by confocal
DIC microscopy. As shown in Figure 4(a), E. coli cells exposed to
these concentrations of C12PB undergo marked morphological
changes when compared with untreated cells: at concentrations
as low as CMC/500, long bacterial filaments, not observed in
647
Inácio et al.
(a)
Control
CMC/100
0.53%
1.86%
0.13%
0.45%
99.34%
97.69%
CMC/80
CMC/50
25.95%
Propidium iodide relative fluorescence intensity
10.61%
2.26%
7.63%
85.31%
60.28%
CMC/30
CMC/20
29.47%
30.52%
9.23%
10.38%
Low doses of QAS impair E. coli normal cell cycle
progression
50.52%
54.88%
CMC/15
CMC/10
40.92%
11.48%
61.67%
37.94%
46.47%
0.16%
SYTO 9 relative fluorescence intensity
CMC/X
(b)
cfu (% of control)
3
1
120
120
100
100
80
80
60
60
40
40
cfu/plate
Propidium iodide
20
0
0.1
1
10
100
C12PB (µM)
20
0
1000 10 000
Viable bacteria (% of control)
10 000 3000 1000 300 100 30 10
Figure 3. Effect of QAS on E. coli membrane integrity. (a) Representative
flow cytometry plots of E. coli cells stained with propidium iodide and
648
control conditions, are visible. Unlike untreated cells, elongated
bacteria do not have a constriction at the centre of the bacterial
body, suggesting a lack of active cell division. At C 12 PB concentrations close to the transition between Processes 3 and 2
(i.e. between CMC/200 and CMC/100), a shrinkage of cells
becomes evident: 80% of the control cells have lengths in the
range 2.4 – 5.0 mm; exposure to C 12 PB at CMC/200 results in
lengths of 1.8 – 3.9 mm; and exposure to C 12 PB at CMC/100
results in lengths of 1.6 – 2.8 mm. E. coli cell length distributions
measured for each experimental condition are shown in
Figure 4(b). Transmission electron microscopy shows that
after incubation with C 12 PB concentrations corresponding to
the toxic Process 3 (i.e. CMC/500), bacteria appear unable
to divide properly, as noticed by the anomalies in the septum
formation and by the elongated morphology that these cells
displayed (Figure 4c). However, structural membrane damage
is only visible at higher C 12 PB concentrations (i.e. CMC/100),
where outer membrane detachment from the cell wall is visible
as an electron-lucent gap between both bacterial cell membranes in addition to shrivelling of the overall structure of the
cell. At CMC/100, cells also display a less electron-dense cytoplasm with occasional emergence of highly electron-dense
clusters, and some leaked contents and debris are detectable
around partially disintegrated cells (asterisk in Figure 4c).
To investigate the first events leading to loss of E. coli culturability
after QAS exposure, the extent of DNA synthesis in individual E. coli
cells was determined by the incorporation of the thymidine
analogue EdU. This method provides a quantitative assay for
DNA replication and allows visual detection of newly synthesized
DNA using fluorescence microscopy. A progressive decrease in the
number of replicating cells was observed after treatment of E. coli
with increasing C12PB concentrations for 1 h (Figure 5a and b). As
can be seen in Figure 5(a), in replicating control cells EdU incorporation after a 15 min pulse yields labelled individual foci, denoting the spatial segregation of newly synthesized DNA from the
replicating sister chromosomes. Moreover, in control cells stained
with a membrane dye, whenever a septum is present it is placed
at the mid cell site and two properly segregated nucleoids are visible. In contrast, after exposure to C12PB for 1 h, EdU foci become
diffuse in elongated cells with abnormal septum formation (i.e.
CMC/500 and CMC/300). At higher concentrations (i.e. CMC/200)
DNA synthesis in those elongated cells seems to stop, as they
SYTO 9 immediately after 1 h of incubation with C12PB. After C12PB
treatment, it is possible to distinguish three different cell populations:
SYTO 9+/propidium iodide 2, intact cells; SYTO 9+/propidium iodide+,
partially damaged cells; and SYTO 92/propidium iodide+, severely
damaged cells. (b) Effect of C12PB on E. coli cell viability as evaluated
either by counting the number of cfu/plate (culture-dependent method)
or using the BacLightTM Bacterial Viability Kit (propidium iodide) that
measures membrane integrity (culture-independent method), after 1 h of
incubation. Data are expressed as percentage of untreated control cultures
and presented as mean+SD of at least three independent experiments.
C12PB concentration is plotted on a logarithmic scale. The CMC of C12PB is
represented by the black dashed line.
JAC
Quaternary ammonium surfactants as microbicides
(b)
CMC/100
CMC/150
CMC/200
CMC/300
* *** *** *** ***
CMC/500
(c)
80
60
40
20
10
8
6
4
2
0
Control
C12PB
Bacterial length (µm)
(a)
Figure 4. QAS induced morphological changes in E. coli cells. (a) Representative confocal DIC images showing E. coli cells after 1 h of incubation with
different concentrations of C12PB. At concentrations where membrane integrity is not compromised (below CMC/100) structures not observed in control
conditions, identifiable as long bacterial filaments, are visible. (b) DIC images were used to estimate the effect of C12PB on E. coli cell length. Data are
presented as box-and-whisker plots. Boxes indicate median and 25th to 75th percentiles while the whiskers represent the lowest and highest values.
Outliers include any experimental point that is more than 1.5 times the IQR. For each sample, at least 100 cells were analysed. Outliers are represented by
the black circles. Kruskal – Wallis test (Dunns’s post-test): *P, 0.05 and ***P, 0.001, significantly different from control. (c) Representative electron
microscopy images showing the differences between the ultrastructure of mock-treated control bacteria and those incubated with C12PB for 1 h.
Morphological changes in the cytoplasmic membrane and failed formation of the septum (compare filled arrowheads in C12PB with control) leads to
the emergence of elongated bacterial cells. At higher concentrations outer membrane detachment is visible as an electron-lucent gap between the inner
and the outer membranes (open arrowhead) and the release of cytoplasmic material is also evident (asterisk).
are no longer EdU positive. Low concentrations of C12PB also exert
a strong effect on nucleoid segregation, since elongated bacteria
were found to contain either a single nucleoid (e.g. CMC/500) or
numerous nucleoids not properly separated from each other
(e.g. CMC/300 and CMC/200). For data on C12TAB and C12BZK
see Figure S3.
QAS effects on E. coli cell cycle progression were further studied
by flow cytometry analysis of cell size and DNA content. In agreement with the microscopy results (Figure 4), after 1 h of incubation with C12PB, the average bacterial size and the distribution
around the peak value (i.e. light scatter) were significantly changed (Figure 5c). At concentrations of CMC/500 and CMC/300, histograms showed a broader distribution of cell size compared with
control, with a higher number of large bacteria, as indicated by a
shift in the right-hand part of the curves. These changes in cell size
distribution were accompanied by a simultaneous increase in
cellular DNA content. At higher concentrations (i.e. CMC/200 and
CMC/150) both the average bacterial cell size and total DNA content decreased (narrower histogram distributions). To evaluate
the extent of DNA replication and bacterial cell cycle status
after incubation with C12PB, cultures were grown in run-out conditions that are routinely employed in E. coli cell cycle assessment.33,34 As shown in Figure 5(c, right panels), after run-out
control cells show integer DNA content consistent with cell
cycle arrestment in the D period (G2-like): the majority of cells
appears to be replicating and arrest at 8N (cells with 8 chromosomes), whereas a smaller subpopulation appears not to have
initiated DNA replication at the time of rifampicin and cefalexin
treatment and arrest at 4N (cells with 4 chromosomes). A small
subpopulation of 2N cells (with 2 chromosomes) is also visible.
In cultures treated with low doses of C12 PB (i.e. CMC/500 and
CMC/300) DNA content is increased. At CMC/300 a shift in the
8N peak occurs, giving rise to a sub-population of cells with
approximately 9 – 10 chromosome equivalents, indicating that
cells are undergoing additional replication rounds without dividing. Our present data cannot exclude the possibility that C12PB
may interact directly with DNA and its processing machinery,
although preliminary results (Â. S. Inácio, T. Ferreira and W. L.
C. Vaz, unpublished data) did not indicate association of the
QAS with DNA in vitro at these low concentrations.
QAS induce changes in membrane potential and cellular
energetics of E. coli
To guarantee a faithful transmission of genetic information to
daughter cells, bacteria possess complex cell division machinery
that tightly synchronizes their chromosome replication and segregation with cell division cycles.67 The regulation of the spatial distribution and correct functioning of cell division-related proteins
requires energy, being highly dependent on the cell metabolic
status68 as well as on the cell membrane potential.69 To test
whether the observed effects of QAS on E. coli cell division were
related to an impairment of cell metabolism, ATP, ADP and AMP
contents of untreated and QAS-treated E. coli were determined.
At concentrations for which no signs of cell membrane damage
were detectable (i.e. CMC/500–CMC/150), C12PB treatment induced
649
Inácio et al.
(a)
EdU
Hoechst
FM4-64
Merged
(c)
Cell size
350
Control
Control
Counts
300
533
150
325
400
100
217
267
50
108
133
0
0
200 400 600 800 1000
CMC/500
Counts
300
325
400
217
267
50
108
CMC/300
Counts
Counts
650
CMC/300
542
325
400
100
217
267
50
108
133
0
0
CMC/200
0
200 400 600 800 1000
650
0
CMC/200
542
200
150
325
400
100
217
267
50
108
133
0
0
CMC/150
300
0
200 400 600 800 1000
650
0
CMC/150
542
533
150
325
400
100
217
267
50
108
FSC-H
4
8
16
CMC/200
4
8
16
CMC/150
133
0
200 400 600 800 1000
CMC/300
667
433
0
16
800
200
0
8
667
533
200 400 600 800 1000
4
800
433
0
CMC/500
667
150
200 400 600 800 1000
16
800
533
300
Counts
200 400 600 800 1000
433
350
Percentage of
EdU-positive cells
0
0
200
0
8
133
0
200 400 600 800 1000
4
667
100
300
100
542
150
350
(b)
CMC/500
533
350
5 µm
0
800
433
0
CMC/150
200 400 600 800 1000
200
0
CMC/200
0
650
Control
667
433
0
CMC/300
800
Control
542
200
350
CMC/500
Chromosome
equivalents
Total DNA content
650
0
200 400 600 800 1000
0
4
8
16
DNA content (SYTO 9) DNA content (SYTO 9)
80
***
60
*** *** ***
40
20
CMC/100
CMC/150
CMC/200
CMC/300
CMC/500
Control
0
Figure 5. C12PB impairs E. coli cell division. (a) Representative fluorescence confocal images showing E. coli cells after 1 h of exposure to C12PB,
co-stained with EdU (proliferating cells), FM4-64 (cell membrane) and Hoechst 33342 (nucleoid). In control cultures, EdU incorporation after a
15 min pulse yields labelled individual foci that represent the spatial segregation of newly synthesized DNA from the replicating sister chromosomes.
In elongated bacterial cells resulting from C12PB treatment, EdU foci become diffuse and at higher concentrations, the elongated bacteria are no longer
EdU positive (arrowheads). (b) Effect of C12PB on E. coli proliferation as evaluated by the incorporation of EdU into DNA after a 15 min pulse. The
percentage of EdU-positive cells was determined by flow cytometry after 1 h of incubation with C12PB. Data are presented as mean+SD of five
independent experiments. One-way repeated measures ANOVA test (Bonferroni’s post-test): ***P,0.001, significantly different from control. (c) Flow
cytometry analysis of cell cycle and cell size of E. coli grown for 1 h in the absence or presence of increasing concentrations of C12PB. Representative
histograms show the distributions of cell size (light scatter, left panels), DNA content (SYTO 9 fluorescence, middle panels) and DNA content after
replication run-out with rifampicin and cefalexin for 4 h (SYTO 9 fluorescence, right panels). In replication run-out conditions, control cultures show
integer DNA content with cells arrested in the D period (G2-like), whereas in cultures treated with low concentrations of C12PB a small population of
cells with more than 8 chromosome equivalents is visible. At concentrations ≥CMC/200 cells display DNA content in an amount consistent with the
B period (G1-like) of the cell cycle. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
650
JAC
Quaternary ammonium surfactants as microbicides
(b)
(d)
150
Counts
120
90
60
Control
CMC/500
CMC/300
CMC/200
CMC/150
30
0
100 101
102 103 104
Rh123
Rh123 relative fluorescence
(fold variation)
Energy charge
0.2
**
6
5
4
3
2
1
0
CMC/100
0.0
CMC/150
C12PB (µM)
***
0.4
CMC/200
50
***
CMC/300
40
0.6
CMC/500
30
**
Control
20
(e)
nmol O2/mg/min
10
***
0.8
CMC/100
0
Control
0
*** ***
***
CMC/150
20
**
CMC/150
40
CMC/200
60
CMC/200
80
1.0
14
12
10
8
6
4
2
0
CMC/300
ATP
ADP
AMP
CMC/500
100
(c)
CMC/300
100
CMC/500
CMC/X
500 300 200 150
ATP/ADP
Percentage of adenylate pool
(a)
180
160
140
120
100
80
60
40
20
0
* **
***
***
NADH
Control
CMC/300
CMC/100
CMC/50
CMC/30
CMC/10
Succinate
Figure 6. C12PB-induced changes in the cellular energetics and membrane potential of E. coli. (a) ATP, ADP and AMP intracellular levels were measured by
reverse-phase HPLC and the relative concentration (%) of adenine nucleotides as a function of the total adenylate pool (ATP+ADP +AMP), (b) the ATP/
ADP ratio and (c) the energy charge were determined after cells were grown for 1 h in the absence or presence of increasing concentrations of C12PB.
Energy charge was calculated as ([ATP] +0.5[ADP])/([ATP]+[ADP]+[AMP]). Data are shown as mean+SD of four independent experiments. One-way
repeated measures ANOVA test (Bonferroni’s post-test): **P, 0.01 and ***P, 0.001, significantly different from control. (d) Representative flow
cytometry histogram showing the effect of increasing concentrations of C12PB on Rh123 uptake by E. coli. The median fluorescence of control
cultures was taken as 1 and the fluorescence of treated samples is expressed relatively to that value (dashed black line). Data are presented as
mean+SD of five independent experiments. One-way repeated measures ANOVA test (Bonferroni’s post-test): **P,0.01, significantly different from
control. (e) Effect of C12PB on E. coli aerobic respiration as assessed by measuring the rate of oxygen consumption of membrane vesicles with an
oxygen electrode. Oxygen uptake was initiated by addition of NADH (1 mM) or succinate (7.5 mM). C12PB was added at the indicated concentrations
5 min prior to vesicle energization. Data are presented as mean+SD of four independent experiments. One-way repeated measures ANOVA test
(Bonferroni’s post-test): *P,0.05, **P,0.01 and ***P, 0.001, significantly different from control. This figure appears in colour in the online version of
JAC and in black and white in the print version of JAC.
a marked decrease in the intracellular ATP content, from
8.7+4.5 pmol/mg of protein in control cells to 0.8+0.4 pmol/mg
of protein at CMC/150 (Figure 6a shows changes in ATP, ADP,
and AMP as a fraction of the total adenylate pool). Concurrently,
the intracellular ADP levels slightly increased (Figure 6a), resulting
in a significant decrease in the ATP/ADP ratio already noticed at
concentrations as low as CMC/500 (Figure 6b). These alterations
were coincident with the appearance of elongated bacteria and
changes in cell proliferation after C12PB exposure. C12PB also
decreased the energy charge of E. coli cells, although statistical
differences from control were only found for concentrations
≥CMC/200 (Figure 6c). The use of the energy charge as an index
of the cell energy status also includes variations in AMP levels.70
The intracellular AMP content remained unchanged after C12PB
treatment: 0.9+0.5 pmol/mg of protein in untreated cells and
0.7+0.4 pmol/mg of protein in cells incubated with a C12PB concentration of CMC/150. However, since the total adenylate pool
(ATP+ADP+AMP) decreased with increasing C12PB concentrations,
at concentrations ≥CMC/200 the relative intracellular AMP content significantly increased (Figure 6a), resulting in a more pronounced reduction of the energy charge. For data on C12TAB
and C12BZK see Figure S4.
QAS effects on bacterial transmembrane potential were
addressed using the membrane potential-sensitive fluorescent
dye Rh123.35 Under our experimental conditions, when cell
membrane structural integrity was not compromised, Rh123 accumulation in the cytoplasm of E. coli cells in response to the electrical
potential across the plasma membrane led to a decrease in fluorescence due to dye self-quenching (Figure S4g). Rh123 accumulation by bacterial cells was uncoupler-sensitive,71 as seen by the
increase in Rh123 fluorescence due to probe efflux after membrane
depolarization with the respiration uncoupler FCCP (Figure S4g).
Membrane depolarization induced by the respiration inhibitor
potassium cyanide72 gave similar qualitative results (Figure S4g).
As shown in Figure 6(d), C12PB reduced the intracellular accumulation of Rh123 in a concentration-dependent manner, resulting in a
651
Inácio et al.
significant increase in fluorescence. In fact, the shift in Rh123 fluorescence induced by a C12PB concentration of CMC/150 was comparable to that of FCCP.
We next evaluated whether the decrease in cell energy charge,
as well as membrane depolarization, induced by QAS could be due
to an inhibition of E. coli respiratory chain activity. Oxygen consumption dynamics were measured with a Clark-type oxygen
electrode in E. coli membrane inverted vesicles using different oxidizable substrates. By using membrane vesicles instead of intact
cells, E. coli respiration can be evaluated independently of ADP
phosphorylation (uncoupled respiration). Figure 6(e) shows the
effect of C12 PB on the respiration rate in E. coli vesicles after
5 min of incubation. Using NADH as an electron donor, oxygen consumption significantly decreased for concentrations ≥CMC/100.
When succinate was used as a substrate, no inhibitory effect of
C12PB on respiration was observed, strongly suggesting that, similar to what happens in mitochondria of mammalian cells,73 QAS
inhibit electron transfer specifically at the level of NADH dehydrogenases (homologue of mitochondrial Complex I). However, contrary to mitochondrial Complex I inhibition, electron transfer
inhibition in E. coli was only observed at concentrations where
membrane integrity was compromised, suggesting that electron
transfer impairment, per se, was not responsible for the altered
bacterial cellular energetics induced by low concentrations of
QAS. Thus, a more plausible explanation for the observed effects
on bacterial membrane potential and intracellular ATP levels may
be that the presence of QAS in the cytoplasmic membrane made
the electrostatic surface charge of the membrane more positive
and, therefore, reduced the effective proton concentration in the
Gouy–Chapman ionic cloud above the membrane surface74 causing the ATP synthase to work at a lower basal rate or in reverse, as
an ATPase, due to the collapse of the proton motive force, leading
to a rapid depletion of the intracellular ATP pool. Strahl and
Hamoen69 recently showed that after membrane depolarization
using an ionophore, E. coli cells displayed an elongated phenotype
similar to what we observed after QAS treatment. The authors
concluded that the transmembrane potential directly modulates
the spatial distribution and organization of several conserved
cytoskeletal and cell division proteins and that membrane
depolarization affected the binding and correct assembly of the
proteins responsible for the Z-ring formation, impeding proper
septum formation. It is, therefore, likely that the appearance of
elongated bacteria after QAS exposure resulted from the blockade
of the septal ring assembly and consequent impaired cytokinesis,
resulting from membrane depolarization and ATP depletion.
In fact, DNA replication was only inhibited at concentrations
at which a more dramatic drop of the intracellular ATP levels
occurred, as can be seen by the increase in the total DNA
amount and EdU incorporation in elongated cells at low C12PB
concentrations.
Conclusions
We have evaluated the acute toxicities of mono-n-alkyl QAS
towards Gram-negative bacteria and human epithelial cells
in vitro with the aim of revealing any discriminatory toxic activity
that could make these compounds useful in the prophylaxis
of STIs and urogenital infections transmitted from motherto-neonate. The impact of two structural properties of QAS was
systematically studied: the length of the apolar n-alkyl group
652
and the chemical nature of the polar head group. Taking into
account each surfactant’s CMC, it was possible to identify a
group of QAS with great potential for prophylactic antisepsis.
Contrary to what is frequently stated in the literature,2 QAS with
short n-alkyl groups with 10–12 carbons were found to be more
efficient and discriminatory microbicides at concentrations that
were sub-toxic for host cells than analogues with 14 –16 carbons.
The discriminatory antimicrobial activity was maximal for analogues with the polar PB head group compared with those with the
TAB or BZK head groups. Therapeutic indices for C12PB are about
≥10 for an MDR E. coli strain and ≥20 for N. gonorrhoeae strains.
Three modes of QAS antibacterial action were identified: (i)
impairment of bacterial energetics and cell division without membrane dissolution or permeabilization at low concentrations, probably due to changes in membrane elastic energy and/or surface
electrostatic properties; (ii) permeabilization, without dissolution
of the bacterial membrane and electron transport inhibition at
somewhat higher concentrations; and (iii) destruction (or dissolution) of the bacterial cell envelope at concentrations close to
the surfactant CMC. On the other hand, distinct mechanisms
underlie QAS toxicity towards eukaryotic cells: QAS inhibit mitochondrial electron transport and oxidative phosphorylation, without causing membrane permeabilization, resulting in apoptotic
cell death.73 Our results suggest that short chain (C12) n-alkyl PB
offer a sufficiently large therapeutic window to merit research on
their use as vaginal microbicides.
We emphasize here that our results relate specifically to acute
toxicity of the QAS to eukaryotic and bacterial cells examined and
do not address the serious problem of development of resistance
by bacteria subject to long-term exposures to biocides.75
Acknowledgements
We thank Dr Célia Nogueira (Microbiology Institute of the Faculty of
Medicine, University of Coimbra, Coimbra, Portugal) for the kind gift of
E. coli API-5144572, Dr Luı́sa Jordão [Instituto Nacional de Saúde Doutor
Ricardo Jorge (INSA), DDI Laboratório de Micobactérias, Lisbon, Portugal]
for performing the biochemical characterization of E. coli API-5144572,
Dr Isabel Nunes Correia (Flow Cytometry Unit, Center for Neurosciences
and Cell Biology, Coimbra, Portugal) for her technical support in the flow
cytometry experiments and Dr Isabel Gordo for her critical comments
on the manuscript prior to submission. We acknowledge Adrian
Velazquez-Campoy and the Institute of Biocomputation and Physics of
Complex Systems (BIFI) at the University of Zaragoza, Spain, for their
help and access to the isothermal titration calorimetry facilities.
Funding
This work was supported by the Foundation for Science and Technology
of the Portuguese Ministry of Science and Higher Education (HMSP-ICT/
0024/2010, PTDC/BIA-BCM/112138/2009 and UID/QUI/00313/2013);
iNOVA4Health-UID/Multi/04462/2013, and the University of Coimbra,
Coimbra, Portugal (Bolsa de Ignição INOV.C 2011), co-funded by the
European Union (FEDER—Fundo Europeu de Desenvolvimento Regional)
through COMPETE—Programa Operacional Factores de Competitividade
and QREN—Quadro de Referência Estratégico Nacional.
Transparency declarations
None to declare.
Quaternary ammonium surfactants as microbicides
Supplementary data
Figures S1 to S4 and Tables S1 and S2 are available as Supplementary data
at JAC Online (http://jac.oxfordjournals.org/).
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