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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. For Permissions, please e-mail: [email protected] 641 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 643 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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