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  • 7/29/2019 Efflux J. Antimicrob. Chemother. 2005 Bredin 1013 5

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    Propyl paraben induces potassium efflux in Escherichia coli

    Jerome Bredin, Anne Davin-Regli and Jean-Marie Pages*

    Enveloppe Bacterienne, Permeabilite et Antibiotiques, IFR 48, Faculte de Medecine,

    Universite de la Mediterranee, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France

    Received 27 October 2004; returned 22 December 2004; revised 15 February 2005; accepted 27 February 2005

    Objectives: Parabens are currently used as antibacterial preservatives in pharmaceutical, cosmetic and

    food products but there are no precise data concerning their activity on bacterial membranes.

    Methods: We analysed the cytoplasmic potassium release during propyl paraben addition by using a

    selective electrode. Various conditions were assayed to investigate the bacterial paraben suscepti-

    bility. We compared the activity of propyl paraben with the activities of colicin A and polymyxin B.

    Results: Propyl paraben induced potassium efflux that was related to the porin expression in the

    bacterial outer membrane. In addition, the presence of spermine, previously described as an efficient

    OmpF channel-blocker, protected susceptible cells against paraben activity.

    Conclusions: Propyl paraben induced potassium release in susceptible Escherichia coli cells similar to

    that observed with polymyxin B. Moreover, this efflux depended on porin channel activity. This

    permeabilizing effect is probably related to antibacterial properties of paraben molecules.

    Keywords: outer membrane proteins, membrane channels, porins, pore forming molecules

    Introduction

    Parabens are commonly added in pharmaceutical, cosmetic and

    food products according to their wide antibacterial properties,

    low toxicity, inertness and chemical stability.1 Although this

    family of molecules is largely used for its antibacterial capacity,

    our knowledge about its activity and bacterial target is quite

    unclear.2 Some authors reported that paraben molecules could

    induce an alteration of cell membrane properties.1,2 Moreover, at

    the moment only few data report the existence of bacterial resist-

    ance against this compound: Valkova et al.3 recently reported

    the characterization of an esterase that is involved in the

    hydrolysis of parabens in Enterobacter cloacae and Enterobacter

    gergoviae. Despite these results, the first steps of parabenactivity, i.e. penetration and targeting, remain quite unclear and

    the aim of this study is to decipher these key points.

    In the present work, we used an in vivo approach based on K+

    efflux measurements as a reporter of membrane alteration after

    paraben addition. We established that propyl paraben alters the

    integrity of bacterial membranes, favouring potassium release,

    and that this mechanism is accelerated by the presence of func-

    tional OmpF porin in the outer membrane. In addition, this pot-

    assium release caused by propyl paraben was compared with the

    effect of colicin A and polymyxin B, two antibacterial agentsthat induce bacterial membrane permeabilization.4

    Materials and methods

    Bacterial strains and susceptibility tests

    The strain used in this work was Escherichia coli SM1005 strain

    (F

    , DlacU169, rpsL, relA, thiA, fibB, gyrA, ompC, ompF14) which

    does not express OmpF or OmpC, and the derivative strain contain-

    ing the plasmid pNLF encoding OmpF.4 Immunodetection assays

    with specific porin antiserum were used to check the level of porin

    synthesis in the strain as previously described. The standard disc

    diffusion method on MuellerHinton (MH) agar was used. Approxi-

    mately 104 cells were inoculated onto plates of MH agar. Variousamounts of propyl paraben or polymyxin B were loaded and the

    corresponding zones of inhibition were measured after 18 h at 378C.

    Evaluation of potassium efflux

    The potassium efflux measurements were carried out after addition

    of propyl paraben, colicin A or polymyxin B as previously

    described.4 Briefly, cells grown to an A600 of 0.6 in Luria-Bertani

    broth were collected by centrifugation and washed with 100mM

    ..........................................................................................................................................................................................................................................................................................................................................................................................................................

    *Corresponding author. Tel: +33-4-91-32-45-87; Fax: +33-4-91-32-46-06; E-mail: [email protected]..........................................................................................................................................................................................................................................................................................................................................................................................................................

    Journal of Antimicrobial Chemotherapy (2005) 55, 10131015

    doi:10.1093/jac/dki110

    Advance Access publication 11 April 2005JAC

    1013

    q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

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

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    sodium phosphate buffer, pH 7. After washing, the pellet was resus-

    pended in 1/100 of culture volume in the same buffer containing 5%

    glycerol. Cells (5 109

    ) were suspended in a glass cup filled with6 mL of the same buffer at 378C and under agitation. Propyl

    paraben was injected when temperature and potassium fluxes were

    equilibrated. Potassium concentration measurements were performed

    with a K+-specific electrode and recorded. Colicin A was obtained

    from our laboratory stock.4 The plotted values, presented as extra-

    cellular K+ increases as previously described,4 are the means of

    three independent experiments.

    Results

    Effect of propyl paraben on bacterial membranes

    Potentiometric measurements performed with an ion-selective

    electrode have been developed to follow membrane permeabili-

    zation in turbid bacterial suspensions; the potassium electrode,

    allowing fast, easy and continuous measurements, is especially

    appropriate for analysing the action of antibacterial compounds

    on the membrane.4,5 To investigate the effect of propyl paraben

    on bacterial membranes, we added various concentrations to

    OmpF-producing E. coli. A major concern with propyl paraben

    solubility is an appropriate medium showing no adverse activity

    on bacterial membranes. We thus tested propyl paraben concen-

    trations up to 0.5 mg/mL. The results are presented in Figure 1(a).

    At this concentration, the addition of propyl paraben induced a

    significant linear K+ release. No lag time was observed after the

    addition of paraben and the curve was quite linear with a slope of

    about 10mM K+

    /min for 0.5 mg/mL, whereas no significantrelease was obtained with lower amounts of propyl paraben.

    It has been previously reported that polymyxin and colicin

    induce an intracellular potassium efflux from E. coli cells.4,5 We

    compared the K+ release induced by propyl paraben with the

    curves resulting from polymyxin B or colicin A addition. Under

    the same conditions, we observed propyl paraben-induced pot-

    assium release kinetics that could be compared to those gener-

    ated by polymyxin B (Figure 1a). These two release kinetics

    contrasted with the kinetics obtained with colicin A. Concerning

    this pore-forming protein, we observed a short lag time followed

    by a linear rapid release, a typical curve of colicin activity, as

    previously reported.4 This suggests that propyl paraben may

    induce a K+ release with a profile and an efficiency close to that

    obtained with polymyxin B.To correlate these results with the antibacterial activity of

    propyl paraben, we measured the inhibition of E. coli growth.

    The results presented in Table 1 indicate that only the porin-

    producing cells were susceptible to propyl paraben under the

    conditions used. In addition, the propyl paraben MIC for this

    OmpF-producing strain was about 1 mg/mL. In contrast, no

    significant change was observed for polymyxin B susceptibility

    whatever the strain tested (Table 1).

    OmpF channel participation in propyl paraben activity

    A major concern with agents that destabilize the membrane

    is the involvement of membrane proteins during the uptake and

    travel to the inner target, e.g. the role of OmpF porin in colicinA activity.4 To clarify this point, we analysed the outer mem-

    brane proteins that can modulate the activity of propyl paraben

    by two independent methods. We measured the potassium leak-

    age in: (i) an OmpF strain and in OmpF-producing cells; and

    (ii) the presence or absence of spermine with the OmpF-produ-

    cing cells. This polyamine has previously been demonstrated to

    jam the OmpF channels and generate a protection against b-

    lactams and fluoroquinolones using this penetration route

    through the outer membrane.6

    Under these conditions, a weak K+ release was observed after

    the addition of propyl paraben to E. coli cells devoid of porin

    while the expression of OmpF porin restored a significant potass-

    ium efflux (Figure 1b). In OmpF-producing bacteria, the K+

    release was severely decreased by the addition of spermine.

    These results indicate that the level of propyl paraben activity

    on the bacterial membrane depended on the presence of func-

    tional porin channels in the outer membrane and are in accord-

    ance with the inhibition measurements.

    Discussion

    The widespread nature of multidrug bacterial resistance mechan-

    isms strongly affects the activity of all antibiotic families.

    Consequently, it is interesting to investigate the effect of propyl

    Figure 1. Effect of propyl paraben on intrabacterial potassium concentration. (a) Measurements of potassium release induced by colicin A (0.1 mg/mL, open

    squares), polymyxin B (0.8 mg/mL, open triangles) and propyl paraben (0.5 mg/mL, open circles; 0.2 mg/mL, crosses). (b) Role of functional OmpF porin in

    paraben activity: experiments were carried out on OmpF cells (open squares), on OmpF+ cells (open circles) and on OmpF+ cells in the presence of spermine

    (1 mM, open triangles) with propyl paraben (0.5 mg/mL). Values were obtained from three independent measurements ( SD).

    Bredin et al.

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    paraben on bacterial membranes since this product is widely

    used as an antimicrobial preservative.1 Despite various studies

    carried out on eukaryotic and prokaryotic cells, there has been

    no clear evidence to explain its antibacterial properties. This is a

    key point since at this moment, several bacterial resistance

    mechanisms against these products have been reported,1,3,7

    including enzymic degradation or efflux pumps.

    In this study, we report that propyl paraben can induce a

    destabilization of bacterial membranes. The profile of potassium

    release is similar to that obtained with polymyxin B, a drug

    inducing outer membrane permeabilization.5 In addition, the

    OmpF porin participates in the activity of propyl paraben. In

    OmpF mutant cells or in the presence of spermine, an efficient

    channel blocker,4 a drastic reduction in potassium release is

    observed. These results are close to those previously reported

    with colicin A: pore-forming colicin uses the OmpF channel to

    penetrate and this process is altered by spermine.4 However, the

    absence of OmpF only reduced the potassium efflux induced bypropyl paraben. The contribution of OmpF porin in propyl para-

    ben activity is especially important taking into account the wide-

    spread use of parabens in cosmetics. This molecule family may

    favour the selection of low-level resistant Gram-negative bac-

    teria with a very low porin expression as previously mentioned

    for cefepime and imipenem, two b-lactam molecules: Gram-

    negative bacteria expressing an altered porin pattern have a

    noticeable level of b-lactam resistance.8 In addition, recent

    studies described that the treatment of E. coli with p-hydroxy-

    benzoic acid (pHBA), a compound structurally related to propyl

    paraben, induced an overexpression of AaeAB, an efflux system

    involved in pHBA resistance.9

    It has been proposed that the cytotoxic action of parabens may

    be linked to mitochondrial failure dependent on induction of

    membrane permeability transition accompanied by the mitochon-

    drial depolarization and depletion of cellular ATP through uncou-

    pling of oxidative phosphorylation.1,2 These observations in

    eukaryotic cells are in accordance with the activity of paraben on

    bacterial membranes reported in this study. The parabens may

    mimic the activity of pore-forming proteins or polymyxins,

    which induce potassium release from the target cell. This potass-

    ium efflux is the first clue of bacterial membrane leakage induced

    by propyl paraben. In this context, it is worth considering the

    paraben content in various standard cosmetic products. Rastogi

    et al.10 have determined that a maximum of 0.32% propyl paraben

    was detected in paraben-containing cosmetics, an amount signifi-

    cantly above the concentration used in this study.

    We can hypothesize that propyl paraben firstly induces the

    permeabilization of bacterial membranes causing the release of

    intracellular molecules and the de-energization of membranes.

    This cascade of processes may support the antibacterial activity

    of this family of preservatives.

    Acknowledgements

    We thank C. Bollet and J. Chevalier for discussions. J. B. was

    funded by the Fondation pour la Recherche Medicale. This study

    was supported by the Universite de la Mediterranee.

    References1. Soni, M. G., Taylor, S. L., Greenberg, N. A. et al. (2002).

    Evaluation of the health aspects of methyl paraben: a review of the

    published literature. Food and Chemical Toxicology40, 133573.

    2. Maillard, J. Y. (2002). Bacterial target sites for biocide action.

    Symposium Series Society for Applied Microbiology31, 16S27S.

    3. Valkova, N., Lepine, F., Labrie, L. et al. (2003). Purification and

    characterization of PrbA, a new esterase from Enterobacter cloacae

    hydrolyzing the esters of 4-hydroxybenzoic acid (parabens). Journal of

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    4. Bredin, J., Simonet, V., Iyer, R. et al. (2003). Colicins, spermine

    and cephalosporins: a competitive interaction with the OmpF eyelet.

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    5. Katsu, T., Nakagawa, H. & Yasuda, K. (2002). Interaction

    between polyamines and bacterial outer membranes as investigatedwith ion-selective electrodes. Antimicrobial Agents and Chemotherapy

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    6. Chevalier, J., Mallea, M. & Pages, J.-M. (2000). Comparative

    aspects of norfloxacin, cefepime and spermine diffusion through the F

    porin channel of Enterobacter cloacae. Biochemical Journal 348,

    2237.

    7. Chuanchuen, R., Karkhoff-Schweizer, R. R. & Schweizer, H. P.

    (2003). High-level triclosan resistance in Pseudomonas aeruginosa is

    solely a result of efflux. American Journal of Infection Control 31,

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    8. Pages, J.-M. (2004). Role of bacterial porins in antibiotic

    susceptibility of Gram-negative bacteria. In Bacterial and Eukaryotic

    Porins (Benz, R., Ed.), pp. 41 59. Wiley-VCH Verlag, Weinheim,

    Germany.

    9. Van Dyk, T. K., Templeton, L. J., Cantera, K. A. et al. (2004).Characterization of the Escherichia coli AaeAB efflux pump: a

    metabolic relief valve? Journal of Bacteriology186, 7196204.

    10. Rastogi, S. C., Schouten, A., de Kruijf, N. et al. (1995). Content

    of methyl-, ethyl-, propyl-, butyl- and benzylparaben cosmetic products.

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    Table 1. Susceptibilities of E. coli to propyl paraben and

    polymyxin B

    Inhibition diameter (mm)

    Compound SM1005 SM1005, pNLF10

    Propyl paraben0.1 mg 6a 6a

    0.5 mg 6a 111 mg 6a 12

    Polymyxin B1 mg 21 225 mg 25 25

    E. coli cells, producing OmpF (SM1005, pNLF10) or devoid of porins(SM1005) were used. Values are means of two independent determinations.aNo inhibition, corresponds to disc/well diameter.

    Propyl paraben alters bacterial membranes

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