Alcohol ethoxylates mediate their bacteriostatic effect by altering the cell membrane of Escherichia coli NCTC 8196

International Journal of Antimicrobial Agents 28 (2006) 503–513

Short communication

Alcohol ethoxylates mediate their bacteriostatic effect by altering the cell membrane of Escherichia coli NCTC 8196 S.L. Moore a , S.P. Denyer b , G.W. Hanlon c,∗ , C.J. Olliff c , A.B. Lansley c , K. Rabone d , M. Jones d a

Reckitt Benckiser, Dansom Lane, Hull, East Yorkshire, UK Welsh School of Pharmacy, Cardiff University, Cardiff, UK School of Pharmacy and Biomolecular Sciences, University of Brighton, Moulsecoomb, Brighton BN2 4GJ, UK d Unilever Research Laboratory, Port Sunlight, Wirral, UK b

c

Received 28 February 2006; accepted 1 August 2006

Abstract The minimum inhibitory concentration (MIC) of a homologous series of alcohol ethoxylates with the same head group size (E6) but differing in the number of carbon atoms in their ‘tail group’ from 10 to 16 was determined for Staphylococcus aureus NCTC 4163 and Escherichia coli NCTC 8196 using a turbidimetric assay. All the surfactants tested demonstrated bacteriostatic activity against both organisms. A tetrazolium assay showed that C14E6 and C16E6 had little effect on the membrane-bound dehydrogenase enzyme activity of E. coli NCTC 8196 compared with C10E6 and C12E6. C10E6 caused leakage both of K+ and nucleotides in a concentration-dependent manner above its MIC of 0.2 mM. C12E6 caused some leakage at concentrations below its MIC (0.12 mM). © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Alcohol ethoxylate; Non-ionic surfactant; Membrane-bound dehydrogenases; Bacterial membrane; Permeability; Escherichia coli; Antimicrobial

1. Introduction Antibacterial agents are composed of a diverse group of molecules and many such agents have entered common use through experience with little information regarding their mode of action. Antibacterial agents can elicit both bacteriostatic and bactericidal effects: bacteriostatic injury has been defined as reversible damage following biocide removal or neutralisation [1], whilst bactericidal action has been described as the irreversible or irreparable damage to a vital cell structure or function [2]. Surfactants have long been known to have considerable antimicrobial activity. However, this knowledge draws mainly from experience with cationic surfactants, most notably the quaternary ammonium salts. Despite being regarded in the past as having little or no antimicrobial effect, the non-ionic surfactants have also been shown to have ∗

Corresponding author. Tel.: +44 1273 642 082; fax: +44 1273 679 333. E-mail address: [email protected] (G.W. Hanlon).

intrinsic antimicrobial activity resulting from their ability to increase the permeability of the bacterial cell membrane. For instance, the non-ionic detergents polyethylene glycol 300, polyethylene glycol 1000 and Lissapol NX were found to cause leakage of cellular material from bacteria, thus demonstrating their ability to alter the permeability of the cell membrane [3]. Schnaitman [4] published a study demonstrating the effects of the non-ionic surfactant Triton X-100, a polyoxyethyloctyl derivative, on Escherichia coli, which showed a solubilising effect of the surfactant on the cell membrane. This appeared to be a specific effect, in that only proteins in the cytoplasmic membrane but not the cell wall were solubilised. In fact, the non-ionic detergents Tween-20 and Triton X-100 were subsequently used to extract an outer membrane-bound protein from the membrane of E. coli in order to allow its purification and characterisation [5]. Lima et al. [6] found that Triton X-100 increased the dissolution of the membrane of Bacillus subtilis and caused ribosome disorganisation. After 15 min of contact, extensive cell lysis was observed. Tween-80, another non-ionic surfactant,

0924-8579/$ – see front matter © 2006 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2006.08.023

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also has membrane-disruptive properties [7]. Lipid vesicles incubated with Tween-80 were found to have increased permeability. Kitatsuji et al. [8] also studied Triton X-100 in addition to Brij 35, another non-ionic surfactant, and found both surfactants to be very effective at lysing filamentous bacteria. These findings imply that Triton X-100 exerts its action by permeabilising the cytoplasmic membrane, as concluded by Lamikanra and Allwood [9] who found that Triton X-100 and Triton X-45 both induced leakage of cytoplasmic constituents, namely potassium ions (K+ ) and material with an absorbance of 260 nm, from Staphylococcus aureus. The amount and rate of K+ leakage from cells exposed to Triton X-45 was found to increase with surfactant concentration. Cserhati et al. [10] investigated the growth inhibitory effect of a homologous series of nonylphenylethylene oxide polymers against Coronilla rhizobium and B. subtilis. They determined that the number of oxide units in the surfactant molecule correlated with the degree to which growth of the two organisms was inhibited. Growth was inhibited by at least 50% in both species when the surfactant molecule contained between 8 and 11 ethylene oxide units. Further work was carried out in the field with the same class of surfactants [11] against a wider range of soil bacteria. Growth of the majority of bacterial species tested was inhibited by the surfactants, with the effect of the surfactants decreasing with increasing length of the hydrophilic ethylene oxide chain. This effect was assumed to be due to membrane damage caused by the bulky nonylphenyl group inserting between apolar fatty acid chains disorganising the membrane structure. Longer ethylene oxide chains modify the distribution of the surfactant between the polar and apolar regions of the membrane with a preference for the aqueous phase, resulting in a decrease or loss of biological activity. The bacterial cytoplasmic membrane is considered to be a major site of action for many surface-active agents. In this study, a range of probes for cytoplasmic membrane damage was used to examine the mechanisms underlying the bacteriostatic properties of a homologous series of non-ionic surfactants. It has been demonstrated previously that 2,3,5triphenyl-tetrazolium bromide can be used to determine the activity of a range of enzymes such as succinate, glucose, mannitol, glycerol and lactate dehydrogenases in E. coli [12]. In addition, the activity of succinate dehydrogenase, which is an integral part of the cell surface of E. coli, has been shown to be almost completely inhibited at concentrations of preservatives that are germicidal to the bacterial suspension under test [13]. In the present study, tetrazolium salts were used as a quantitative marker of damage to the membranebound dehydrogenase enzymes succinate dehydrogenase and glucose dehydrogenase. Further changes to the cytoplasmic membrane of bacterial cells can be followed by leakage of cellular constituents such as K+ and/or pentose sugars. Leakage of intracellular K+ has been used as an indicator of damage to membrane integrity of the bacterial cell following addition of biocide [14] and therefore allows assessment of biocidal capability. Since the

K+ ion is very small and leakage has been found to occur very rapidly after contact with an antibacterial agent, K+ release is regarded as an effective marker of subtle alterations to the permeability of the cytoplasmic membrane. To evaluate more extensive membrane damage, monitoring of larger molecules such as nucleotides is suitable: leakage of nucleotides and their constituents (pyrimidines and purines) in the presence of membrane-active compounds can be observed by absorbance measurements (at 260 nm) of cellfree exudates. Such leakage is dependent not only on biocide concentration but also on the organism itself, its metabolic pool and the conditions under which leakage is being studied. At physiological temperatures (35–40 ◦ C), a rapid release of material with an absorbance of 260 nm occurs, which is followed by a more gradual leakage [15–18]. It has been proposed that this secondary leakage is due to the activation of latent ribonucleases, which break down ribosomal RNA [19]. Generally, the physicochemical properties of compounds have a marked influence on their biological activity [20]. For instance, the critical micelle concentration (CMC) of a compound has been shown to be correlated with antibacterial properties. Cella et al. [21] demonstrated that decreasing CMC values correlated with germicidal activity, whilst Lein and Perrin [22] reported increased protein binding ability with decreasing CMC values. In addition, log P, a measure of the lipophilicity of a compound, also displays a relationship with biological activity. Gilbert [23] described a parabolic relationship when log P was plotted against biological activity for a homologous series of compounds. The optimum log P values for Gram-negative and Gram-positive cells have been determined as 4.0 and 6.0, respectively [24]. In this study, the antibacterial activity of a class of nonionic surfactants, the alcohol ethoxylates, was determined. By evaluating the ability of the compounds to alter the properties of the cytoplasmic membrane and by monitoring the leakage of cytoplasmic constituents from E. coli NCTC 8196, the mechanisms of action of the surfactants were explored with reference to some of their physicochemical properties such as their CMC, molecular mass and lipophilicity.

2. Materials and methods 2.1. Determination of minimum inhibitory concentration (MIC) The MIC of the surfactants was determined for S. aureus NCTC 4163 and E. coli NCTC 8196 (both obtained from the National Collection of Type Cultures, Colindale, London, UK) after 6 h and 24 h contact time. Stock solutions of the surfactants were prepared in distilled water and then filtersterilised using a 0.2 ␮m cellulose nitrate membrane filter (Nalgene, Hereford, UK). Any potential sorption sites on the syringe and filter were saturated by pre-rinsing the syringe with the surfactant solution and discarding the first 5 mL

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of solution filtered through the membrane. Dilutions of surfactant solutions (on an arithmetic scale from 0.01–10 mM, depending on the surfactant tested) were prepared in synthetic broth AOAC (Difco Laboratories, Oxford, UK). An aliquot (200 ␮L) of each concentration of surfactant solution was placed in the wells of a 96-well microtitre plate each containing 50 ␮L of a suspension of 5 × 106 bacterial cells/mL (grown and prepared in synthetic broth AOAC), giving a final cell concentration of 1 × 106 cells/mL. Dilutions of surfactant solutions in the absence of bacteria and of bacteria in the absence of surfactant were also prepared as negative and positive controls, respectively. The microtitre plate was then incubated at 37 ◦ C. Wells were examined for turbidity (as an indication of growth) at 6 h and 24 h. Each determination was performed twice, with the exception of the anionic surfactant sodium dodecyl sulphate (SDS) and E. coli NCTC 8196 at 24 h, which was performed six times to determine the reproducibility of the method. The MIC, which is defined as the lowest concentration at which there is no visible turbidity, was unchanged for each replicate. The MICs of the surfactants penta(ethyleneglycol)-monododecyl ether (C10E6), hexa(ethyleneglycol)-monododecyl ether (C12E6), septa(ethyleneglycol)-monododecyl ether (C14E6) and octa(ethyleneglycol)-monododecyl ether (C16E6) in the homologous series of alcohol ethoxylates were determined after 6 h incubation. These surfactants were provided by Unilever Research (Port Sunlight, UK). 2.2. Determination of CMC and log P The CMCs for the homologous series of alcohol ethoxylates (C10E6, C12E6, C14E6 and C16E6) were determined by a dye solubilisation method [25,26]. The method is based on the observation that solubilisation of a hydrophobic dye such as Orange OT in a detergent solution only occurs if micelles are present. An aliquot (300 ␮L) of 0.1% w/v Orange OT (SigmaAldrich, Dorset, UK) in ethanol was pipetted into each well of a 48-well microtitre plate. The ethanol was allowed to evaporate leaving a film of dye on the bottom of the wells. Increasing concentrations of surfactant (0.002–10 mM) both in synthetic broth AOAC and in McIlvaine’s buffer (pH 7.0) (1.2 mL) were then added to the wells and the plate was agitated for 1 min on an orbital shaker. The plate was then incubated for 18 h at 37 ◦ C. After this period of incubation, the samples were transferred to a fresh plate for spectrophotometric analysis. The absorbance of the samples was read at 492 nm (Lambda 2 spectrophotometer; PerkinElmer, Boston, MA) against a blank of either synthetic broth AOAC or McIlvaine’s buffer (pH 7.0) that had been previously incubated with the dye under the same conditions as the samples. McIlvaine’s buffer (100 mL) was prepared by adding 18.15 mL of citric acid (0.1 M) to 81.85 mL of disodium hydrogen orthophosphate 2-hydrate (0.2 M) and adjusting the pH using hydrochloric acid or sodium hydroxide (all ingredients from BDH Ltd., Dorset, UK).

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Data were plotted as surfactant concentration against absorbance (not shown), giving a clear discontinuity in the relationship. Linear regression analysis was used to determine accurately the intersection point between the two lines of best fit; this inflection point was taken as the CMC of the surfactant. The log P values of the homologous series of surfactants, where P is the octanol/water partition coefficient, were calculated using the method of Hansch and Leo [27]. 2.3. Formazan production Cultures of E. coli NCTC 8196 were grown for 15 h at 37 ◦ C on Roux slopes prepared from synthetic broth AOAC solidified with 1.5% w/v technical grade agar No. 1 (Oxoid Ltd., Basingstoke, UK). Bacterial cells were harvested by washing the surface of the slope with 40 mL of McIlvaine’s buffer (pH 7.0). The cell suspension was centrifuged at 2100 × g for 20 min and then washed twice with McIlvaine’s buffer (25 mL). A series of bijou bottles (volume 5 mL) was prepared containing 1 mL of glucose (0.02 M) or sodium succinate (0.02 M) (1 mL) in McIlvaine’s buffer, 2,3,5triphenyltetrazolium chloride (0.05% w/v) in McIlvaine’s buffer (1 mL) and alcohol ethoxylate (C10E6, C12E6, C14E6 or C16E6), giving final concentrations from 0.005 mM to 5 mM in McIlvaine’s buffer (2.5 mL). Each bottle represented a single time point for the experiment. The bottles were covered to prevent exposure to light and then incubated at 37 ◦ C prior to the start of the experiment to enable the temperature of the solutions to equilibrate. The experiment was initiated by the addition of 0.5 mL of an E. coli NCTC 8196 cell suspension to give a final concentration of 1 × 109 cells/mL and the bottle was inverted to ensure even distribution of the bacteria. Control samples were prepared with the surfactant/buffer solution replaced by buffer alone. Samples were incubated statically at 37 ◦ C over a 60-min period and at appropriate time intervals the contents of the bijou bottles were vortexed with 5 mL of ethyl acetate and 1 mL of NaOH (1 M) to extract the formazan into the ethyl acetate. The samples were then centrifuged for 5 min at 2100 × g to reduce the emulsion formed during the extraction procedure. Absorbance of the samples of extracted formazan in ethyl acetate was then measured at 450 nm (Perkin-Elmer Lambda 2 spectrophotometer) against a blank of an ethyl acetate extract of bacteria, glucose and McIlvaine’s buffer. 2.4. K+ leakage Cultures of E. coli NCTC 8196 were grown for 15 h at 37 ◦ C on Roux slopes composed of synthetic broth AOAC solidified with 1.5% w/v agar supplemented with 0.1% w/v potassium chloride to raise the intracellular K+ concentration. The bacteria were harvested by washing the surface of the slope with 100 mL of magnesium chloride solution (0.1 M) (AnalaR; BDH, Poole, UK). The cell suspension was cen-

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trifuged at 2100 × g for 20 min and the pellet was washed twice with 25 mL of magnesium chloride solution (0.1 M). The washed cell pellet was standardised to give a cell concentration of 1 × 1010 cells/mL in McIlvaine’s buffer (pH 7.0), which is potassium-free. To determine K+ leakage induced by surfactant, 2 mL aliquots of the E. coli NCTC 8196 cell suspension (1 × 1010 cells/mL) were added to 18 mL of McIlvaine’s buffer containing either C10E6 (to give final concentrations of 0.025, 0.05, 0.2, 0.5 and 1 mM) or C12E6 (to give final concentrations of 0.005, 0.05, 0.2 and 0.5 mM). Samples (2 mL) were removed after 2.5, 5, 10, 20, 30, 45 and 60 min incubation and filtered through a 0.2 ␮m cellulose nitrate membrane filter to remove the bacteria. The filtrate was frozen at −20 ◦ C. Surfactantfree controls were prepared to determine normal K+ flux over the time course of the experiment. Prior to analysis, the samples were thawed and then diluted 1 in 5 with McIlvaine’s buffer before being studied for K+ content using an atomic absorption spectrophotometer (M1100 Atomic Absorption Spectrophotometer; Perkin-Elmer, Boston, MA) in the flame emission mode. To determine the intracellular K+ pool, 2 mL aliquots of the E. coli NCTC 8196 cell suspension (1 × 1010 cells/mL) were added to 18 mL of pre-warmed (37 ◦ C) McIlvaine’s buffer containing 1 mM cetrimide (Sigma-Aldrich, Dorset, UK). The sample was mixed thoroughly and incubated at 37 ◦ C for 10 min. Six samples (2 mL) were then withdrawn and filtered through a 0.2 ␮m cellulose nitrate membrane filter to remove the bacteria and the filtrate was frozen at −20 ◦ C until analysis for K+ content. 2.5. Leakage of material with an absorbance of 260 nm Escherichia coli NCTC 8196 cells were cultured and harvested as previously described and reconstituted to yield a suspension containing 1 × 1010 cells/mL in McIlvaine’s buffer. To determine the total amount of intracellular material with an absorbance of 260 nm, i.e. soluble material constituting the metabolic pool and insoluble material from the autolysis of nucleic acids, the trichloroacetic acid (TCA) method of Gale and Folkes [28] was used. A volume (10 mL) of E. coli NCTC 8196 cell suspension (1 × 109 cells/mL) in 5% w/v TCA was incubated for 2 h at 20 ◦ C. This process extracts the soluble metabolic pool of material absorbing at 260 nm. The resulting cell suspension was then centrifuged for 15 min at 2100 × g and the supernatant liquid was filtered through a 0.2 ␮m cellulose nitrate membrane filter. The remaining cell pellet was then extracted a further three times with 5% w/v hot TCA at 100 ◦ C for 10 min and filtered as described previously. Three extractions with hot TCA were found by Gale and Folkes [28] to be sufficient to release all the material absorbing at 260 nm from the cells. The absorbance of all the extracts was measured at 260 nm against a 5% w/v TCA blank. To determine the effect of alcohol ethoxylates on the leakage of material with an absorbance of 260 nm from E. coli

NCTC 8196, 2 mL aliquots of the cell suspension (1 × 1010 cells/mL) were added to 18 mL of McIlvaine’s buffer prewarmed to 37 ◦ C containing either C10E6 (to give final concentrations of 0.025, 0.05, 0.2, 0.5 and 1 mM), C12E6 (to give final concentrations of 0.005, 0.05, 0.2 and 0.5 mM) or neither surfactant. The cell suspensions (final concentration 1 × 109 cells/mL) were then incubated at 37 ◦ C and a 2 mL sample of the reaction mixture was removed with a syringe at 5, 10, 20, 30, 45, 60, 120 and 240 min and filtered through a 0.2 ␮m cellulose nitrate membrane filter. The samples were frozen at −20 ◦ C until analysis. The absorbance at 260 nm of the samples was measured (Perkin-Elmer Lambda 2 Spectrophotometer) against a blank solution containing the appropriate concentration of C10E6 or C12E6 and diluted with McIlvaine’s buffer to the same degree as the samples.

3. Results All of the surfactants exhibited antibacterial activity and in general they were more effective against the Gram-positive bacterium S. aureus NCTC 4163 than against the Gramnegative bacterium E. coli NCTC 8196 (Table 1). The nonionic alcohol ethoxylates possessed greater antimicrobial activity than SDS at 6 h. The head group size of the alcohol ethoxylates, with the exception of C12E8, appeared to have little influence on the antimicrobial activity. To determine whether varying the alkyl chain length of the alcohol ethoxylates had any significant effect, a homologous series sharing the E6 head group with alkyl chains from 10 to 16 carbons was investigated for antimicrobial activity. Since E. coli NCTC 8196 demonstrated greater resistance to the alcohol ethoxylates than S. aureus NCTC 4163, this organism was considered to offer the best opportunity for discriminating between agents within the series and was therefore selected for further study.

Table 1 Minimum inhibitory concentrations (MICs) of various non-ionic surfactants and sodium dodecyl sulphate (SDS) Surfactantsa

SDS C12E4 C12E5 C12E6 C12E7 C12E8 C10E6 C12E6 C14E6 C16E6 a b

MIC (mM)b E. coli (6 h)

E. coli (24 h)

S. aureus (6 h)

S. aureus (24 h)

0.28 0.11 0.10 0.12 0.08 0.30 0.20 0.12 5.0 5.0

0.55 >0.58 >0.58 >0.58 >0.58 >0.58

0.28 <0.03 <0.03 <0.03 <0.03 <0.03

0.55 0.11 0.10 0.09 0.08 0.15

n = 2 except SDS where n = 6. The MIC was unchanged for each replicate.

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Table 2 Molecular mass, calculated log P and critical micelle concentrations (CMCs)a of the homologous series of alcohol ethoxylates Alcohol ethoxylate

C10E6 C12E6 C14E6 C16E6

Molecular mass

422.6 450.7 478.7 506.8

Log P

3.65 4.73 5.81 6.89

CMC (mM) McIlvaine’s buffer (37 ◦ C) (n = 6)

Broth (37 ◦ C) (n = 6)

Water (25 ◦ C)

0.653 0.189 0.170 0.082

0.908 0.344 0.258 0.222

0.9 0.087 0.01 0.0017

a CMCs are given both in McIlvaine’s buffer (pH 7.0) and in synthetic broth AOAC, and for reference are compared with values determined in water by surface tension measurements [29].

The CMCs for the homologous series of alcohol ethoxylates both in McIlvaine’s buffer and in synthetic broth AOAC are presented in Table 2. They can be compared with those determined in water by surface tension measurements at 25 ◦ C [29]. It can be seen from the values that as the length of the alkyl chain increases, the CMC decreases whilst lipophilicity increases. The relationship between log P and biological activity (1/MIC) for the homologous series of alcohol ethoxylates was also examined (Fig. 1). The relationship is not linear, with a significant discontinuity between C12E6 and C14E6; a peak in activity is seen for C12E6, which has a log P value of 4.73. Significant inhibition (>50%) of formazan production by membrane-bound succinate and glucose dehydrogenase enzymes was observed at concentrations of C10E6 of ≥0.20 mM after 10 min (P < 0.001, analysis of variance (ANOVA)). Below this concentration no significant inhibition was observed even after 60 min exposure of E. coli NCTC 8196 to surfactant (P > 0.05) (Fig. 2). A concentrationdependent effect on formazan production was also observed in the presence of C12E6 (Fig. 3). With glucose as the substrate, formazan production was inhibited at concentrations of ≥0.05 mM (P < 0.001) within 10 min (Fig. 3a); at 0.025 mM, C12E6 inhibited formazan production within 20 min (P < 0.001), which was >50% after 30 min. Interestingly, with the succinate substrate, formazan production was not inhib-

Fig. 1. Relationship between biological activity and log P for a homologous series of alcohol ethoxylates. MIC, minimum inhibitory concentration.

ited at a concentration of 0.05 mM C12E6 even after 60 min and inhibition was not observed until the bacterial cells were exposed to a concentration above the MIC of 0.12 mM C12E6 (Fig. 3b). The lower concentrations of C14E6 (0.05 mM and 0.1 mM) did not inhibit formazan production with glucose as the substrate (P > 0.05). However, significant inhibition of formazan production was observed for the higher concentrations of C14E6 (0.3 mM and 0.5 mM) after as little as 10 min (P < 0.001) (Fig. 4a). With succinate as the substrate, con-

Fig. 2. Formazan production (mean ± standard deviation; n = 6) by Escherichia coli NCTC 8196 with substrates of (a) glucose and (b) succinate in the presence of varying concentrations of C10E6.

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Fig. 3. Formazan production (mean ± standard deviation; n = 6) by Escherichia coli NCTC 8196 with substrates of (a) glucose and (b) succinate in the presence of varying concentrations of C12E6.

centrations of 5.0 mM C14E6 inhibited formazan production by ca. 50% after 10 min contact (P < 0.001). This degree of inhibition is similar to that exerted by 0.5 mM C14E6 on formazan production using a glucose substrate. With succinate as the substrate, 0.5 mM C14E6 only inhibited formazan production after an extended contact time (ca. 50 min). Indeed, at earlier time points there appears to be a significant stimulation of formazan production with 0.05 mM and 0.2 mM C14E6 (Fig. 4b) (P < 0.001). With glucose as the substrate, C12E6 inhibits formazan production at a concentration that is an order of magnitude less than that of either C10E6 or C14E6, which have similar potency (P < 0.001). Interestingly, with succinate as the substrate, equimolar concentrations of C10E6 and C12E6 inhibit formazan production and these two surfactants are more potent than C14E6. When considering the effect of these surfactants at a concentration of 0.5 mM with succinate as the substrate, both C10E6 and C12E6 inhibit formazan production significantly more than C14E6 (P < 0.001). C16E6 did not inhibit formazan production with either glucose or succinate as the substrate at any of the con-

Fig. 4. Formazan production (mean ± standard deviation; n = 6) by Escherichia coli NCTC 8196 with substrates of (a) glucose and (b) succinate in the presence of varying concentrations of C14E6.

centrations tested (0.05–5 mM) at any exposure time (data not shown). Determination of the total intracellular K+ pool yielded a mean figure of 196.89 ± 11.88 ␮M (n = 6). K+ leakage from control cells (56 ± 6 ␮M) was shown to occur and was thought to be a consequence of the harvesting and washing procedure despite the use of magnesium chloride, which helps to protect cells from various stresses [30]. However, leakage over and above that seen with the controls was induced in the presence of higher concentrations of alcohol ethoxylates and control values were subtracted from all test results. Release profiles over time for C10E6 and C12E6 showed rapid leakage of K+ at higher concentrations, which generally levelled off at ca. 10 min. For clarity, the data given in Fig. 5 are for a single representative time point of 20 min. At the lowest concentration of C10E6 (0.025 mM), K+ leakage was significantly lower than that seen with the control (P < 0.01), suggesting some sort of sealing-in process. This was also the case with the lowest concentration of C12E6 (0.005 mM) for contact times in excess of 20 min. K+ leakage

S.L. Moore et al. / International Journal of Antimicrobial Agents 28 (2006) 503–513

Fig. 5. Potassium (K+ ) leakage (mean ± standard error of the mean; n = 6) from Escherichia coli NCTC 8196 after 20 min exposure to increasing concentrations of C10E6 and C12E6. Leakage of K+ from control cells (56.00 ± 6.00 ␮M) (n = 6) has been subtracted from all values of K+ concentration. Intracellular K+ pool = 196.89 ± 11.88 ␮M (n = 6).

occurred in a concentration-dependent manner in the presence of C10E6 (Fig. 5). Significant K+ leakage occurred at the MIC of 0.20 mM (P < 0.01) and above: no significant leakage (P > 0.01) in excess of the control was seen below this concentration of C10E6. The greatest K+ leakage was recorded at a concentration of 0.5 mM, which approximates to the CMC for C10E6 (0.653 mM) in McIlvaine’s buffer at 37 ◦ C. With C12E6, the effects were similar but less pronounced. In addition, a concentration of 0.2 mM generally gave a greater level of K+ leakage than 0.5 mM. This value for maximal leakage again approximates to the CMC for C12E6 (0.189 mM). Using the cold TCA method, the amount of material extracted from the bacterial cells gave an absorbance at 260 nm of 0.676, which represented the total soluble metabolic pool of material. The remaining insoluble cellular material was extracted using hot TCA and gave an absorbance of 2.087. Thus, the total absorbance of all cellular material was 2.763. Leakage of 260 nm-absorbing material was found to occur from control cells not exposed to surfactant. This value (0.7 ± 0.12 absorbance units) was subtracted from all test samples recorded. Release profiles for 260 nmabsorbing material demonstrated a steady increase in leakage up to 60 min, at which point the effect reached a plateau (data not shown). For clarity, the data given in Fig. 6 show results at a representative time point of 60 min. In the presence of higher concentrations of surfactant, the release of 260 nm-absorbing material from the bacterial cells indicated that both soluble and structural nucleic acid material was being released. The presence of the lowest concentration of C12E6 appeared to cause a reduced level of leakage compared with that seen with the control, although this was not significant (P > 0.05). Leakage of 260 nm-absorbing material was not observed at concentrations below the MIC (0.20 mM) for C10E6, whilst for C12E6 release was seen at all but the lowest concentration.

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Fig. 6. Leakage of material with an absorbance of 260 nm (mean ± standard error of the mean; n = 6) from Escherichia coli NCTC 8196 after 60 min exposure to increasing concentrations of C10E6 and C12E6. The absorbance at 260 nm of material leaking from control cells (0.70 ± 0.12 absorbance units) has been subtracted from all absorbance values.

Where significant release of material with an absorbance of 260 nm occurred, this was in excess of the metabolic pool, indicating that nucleic acids were being broken down and therefore autolysis was occurring. Comparing equivalent concentrations of C10E6 and C12E6, both compounds appeared to be equally effective at causing the release of material with an absorbance of 260 nm.

4. Discussion The studies reported here demonstrate that all of the alcohol ethoxylate surfactants tested had some degree of bacteriostatic action against either E. coli NCTC 8196 or S. aureus NCTC 4163. In general, the surfactants were more effective against the Gram-positive S. aureus NCTC 4163 than against the Gram-negative E. coli NCTC 8196. In comparison, the anionic surfactant SDS did not appear to discriminate. Similar results regarding the differing susceptibilities of Grampositive and Gram-negative organisms to anionic surfactants have been reported previously [31,32]. Such differences are usually attributed to the differences in the cell wall. The relationship between biological activity and various physicochemical characteristics can be complex. Examination of the effect of increasing molecular weight on the bacteriostatic effect of the surfactants showed that the head group size of the ethoxylates appeared to have little influence on antimicrobial activity, with the exception of C12E8 (Table 1). However, a discriminating relationship between MIC and chain length was observed for the homologous series of alcohol ethoxylates. Using MIC as a measure of biological activity, it can be seen that C10E6 and C12E6 have a much higher biological activity than C14E6 and C16E6. The increased biological activity of C10E6 and C12E6 may be due to the fact that their log P values (3.65 and 4.73, respectively)

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are close to the optimum value of 4.0 for Gram-negative bacteria [24]. In contrast to the results of the current study, a linear relationship between chain length and activity has been reported previously for a series of non-ionic polyoxyethylene-type surfactants [33], which agreed with the work of Zaslavsky et al. [34], i.e. the higher the molecular weight the less active the compound. A similar linear relationship was observed between partition coefficient and activity of octyl phenols [35]. Optimum values for biological activity have also been reported by Kabara [36] and Devinsky et al. [37], which support the results of the current study. The CMC values obtained for the alcohol ethoxylates decreased as the alkyl chain length increased, as has been reported previously [38]. There is some correlation between the MIC and CMC of the homologous series; the MIC is inversely proportional to the CMC value obtained, with the exception of C12E6, which possesses a lower MIC than that of C10E6. Previous work determining the effect of a range of anionic and non-ionic surfactants against bacteria suggested the site of damage for the surfactants to be the cytoplasmic membrane [3,4,6,39–42]. Thus, damage to this site was examined in the current study using a range of biochemical approaches. Inhibition of membrane-bound dehydrogenase enzymes was concentration-dependent for C10E6 and was observed at concentrations of 0.2 mM and above (MIC = 0.20 mM) both with the glucose and succinate substrates. The inhibitory concentrations were well below the CMC of C10E6 (0.653 mM in McIlvaine’s buffer). Concentrations above the CMC were not examined. Inhibition of dehydrogenase activity occurred at concentrations of C12E6 of ≥0.025 mM with the glucose substrate. The lower concentrations were well below the MIC (0.12 mM) and CMC (0.189 mM in McIlvaine’s buffer). Only the effect of one concentration of C12E6 was examined, which was considerably greater than the CMC; interestingly, this concentration (0.5 mM) showed a similar degree of toxicity as 0.2 mM C12E6. This suggests that it is the monomer of the surfactant that is active. With C14E6, inhibition of dehydrogenase enzymes was observed at concentrations above its CMC (0.170 mM in McIlvaine’s buffer) with the glucose substrate; however, there was not the concentration-dependent effect seen with C10E6 and C12E6. The lack of concentration dependence is likely to reflect the fact that most of the concentrations used were above the CMC of C14E6. This reinforces the idea that it is the monomers that are active. All the concentrations used were below or at the MIC of C14E6 (5 mM). C16E6 had little effect on formazan production and almost all the concentrations studied exceeded the CMC of C16E6 (0.082 mM in McIlvaine’s buffer). This further suggests that formation of micelles diminishes the activity of the surfactants. Stimulation of dehydrogenase activity seen at low concentrations and early time points for some surfactants has also been reported by Hugo and Longworth [43] and Gilbert et al. [44] with chlorhexidine and phenoxyethanol, respectively.

This stimulation has been suggested to be an indirect effect of the compounds uncoupling oxidative phosphorylation [45]. At the later time points, significant inhibition is seen with higher surfactant concentrations, particularly in the presence of C10E6 and C12E6. This suggests that there is damage to membrane-bound dehydrogenase enzyme function, possibly through disturbance of the respiratory chain. KopeckaLeitmanova et al. [46] determined dehydrogenase activity in the presence of amine oxides and quaternary ammonium salts and also observed differences in the effect of low and high concentrations. This group found that inhibition was similar whether glucose or succinate was used as the substrate and further proposed that the surfactants did not specifically affect the dehydrogenase enzymes but that the effects observed were a secondary consequence of primary physical damage. In the present study, the pattern of inhibition with glucose and succinate was not the same for both surfactants. C10E6 showed similar inhibition in the presence of either substrate, whilst C12E6 demonstrated greatly reduced formazan production in the presence of glucose compared with that seen with succinate. This suggests there could be a subtle difference in the effect of these two surfactants on the respiratory chain. Succinate dehydrogenase is closely associated with the cytoplasmic membrane, which is the site of surfactant action, whereas glycolysis proceeds in the cytoplasm. The inhibition of glucose uptake in the presence of C10E6 and C12E6 is minimal, indicating that the effect on respiratory chain activity is not caused by reduced glucose uptake (data not shown). The outer membrane of Gram-negative bacteria such as E. coli contains porins that have an aqueous environment and favour the passage of hydrophilic molecules with a molecular weight less than 600 Da [47]. Although all members of the homologous series of alcohol ethoxylates had a molecular weight of <600 Da, there was significant variation in the hydrophilicity of the four compounds. The two shorter chain length members of the series (C10E6 and C12E6) were relatively hydrophilic in character (log P values of 3.65 and 4.73, respectively), whilst C14E6 and C16E6 were more lipophilic (log P values of 5.81 and 6.89, respectively) and therefore less likely to cross the outer membrane of the bacterial cell via the porins. In addition, C14E6 and C16E6 both form micelles at lower concentrations than C10E6 and C12E6, which would further limit movement through the pores. This may explain the increased antimicrobial activity of C10E6 and C12E6 against E. coli NCTC 8196 in comparison with C14E6 and C16E6, since the two former compounds probably reach the cytoplasmic membrane of the bacterial cell in higher concentrations than the latter two. Later work therefore concentrated on C10E6 and C12E6. Both C10E6 and C12E6 altered the permeability of the cytoplasmic membrane of E. coli NCTC 8196 causing leakage of cytoplasmic constituents. The toxicity of the two surfactants was similar at similar concentrations. Low levels of both surfactants appeared to reduce leakage both of 260 nm-absorbing material and K+ below that of the control;

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this could be due to a ‘sealing-in’ effect [48]. In this situation, low concentrations of surfactant are thought to protect the membrane from leakage, which may be due to surfactant monomers introducing themselves into the cytoplasmic membrane thus reducing the fluidity of the membrane and therefore leakage. As the concentration of surfactant in the membrane increases, so too would disruption to the membrane structure and therefore the cytoplasmic constituents would begin to leak out. In a study of the effect of the nonionic detergent octyl glucoside on membrane permeability, a model was developed which stated that if the effective crosssectional areas of the phospholipid molecules in the membrane and the detergent molecules are similar to one another, a detergent molecule in the membrane phase will be surrounded only by phospholipid molecules as long as the mole fraction of the detergent in the membrane phases is below 0.3; under these conditions, membrane barrier efficiency is high. At a mole fraction higher than 0.3, the detergent molecules come into contact with each other and the membrane barrier efficiency decreases [49]. The membrane-sealing effect of the non-ionic surfactant Poloxamer 188 (0.1 mM) has been demonstrated by adding it to the medium of cultured irradiated mammalian cells and inhibiting leakage from the cells that would otherwise have been expected to occur post irradiation [50]. K+ and 260 nm-absorbing material leakage from E. coli NCTC 8196 in the presence of C10E6 occurred in a concentration-dependent manner, as observed by Lamikanra [51] who found that treatment of S. aureus and E. coli with butyl hydroxyanisole caused leakage of intracellular materials, with leakage increasing as biocide concentration increased. The CMCs of C10E6 and C12E6 in McIlvaine’s buffer (pH 7.0) at 37 ◦ C are 0.653 mM and 0.189 mM, respectively. Thus, all the concentrations of C10E6 used in the study were below the CMC and the number of surfactant monomers in solution would be expected to be proportional to the concentration of surfactant employed. However, 0.5 mM C12E6 is above the CMC of this surfactant and an equilibrium between the number of surfactant monomers in solution and present in the micelles would be expected to exist, reducing the number of monomers available for interaction with the bacterial cell membrane and explaining why 0.5 mM C12E6 was less damaging to bacterial cells than 0.2 mM C12E6. The results of the present study show that ca. 50% of the K+ ions are released from the cells within the first 5 min of contact. This early leakage has been seen by other workers [52–54]. As contact time with the surfactants lengthens, material with an absorbance of 260 nm is increasingly released. This could be due to progressive membrane damage. High levels of 260 nm-absorbing material released in excess of that from the metabolic pool are due to the autolytic breakdown of insoluble nucleic acid [18]. The chemiosmotic theory [55,56] proposed that production of ATP by the cell was a consequence of the proton gradient across the membrane of the cell, as are some transport processes. If this gradient is disturbed, as it must be when

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the permeability of the membrane is disrupted sufficiently to allow leakage of cellular constituents, then these processes will be affected. Therefore, the effect of C10E6 and C12E6 on the permeability of the cytoplasmic membrane may cause inhibition of ATP-ase and glucose uptake, ultimately contributing to poor cell recovery. A simulation method, namely dissipative particle dynamics, has been applied to investigate the cause of cell death when bacteria are exposed to non-ionic surfactants [57]. Mixed bilayers of lipid and non-ionic surfactant were studied and the diffusion of water through the bilayer was monitored. Small transient holes were seen to appear at 40% mole-fraction C9E8, which became permanent holes between 60–70% surfactant. When C12E6 was applied, permanent holes only arose at 90% mole-fraction surfactant. Some simulations were carried out to determine the rupture properties of mixed bilayers of phosphatidylethanolamine and C12E6. These indicated that the area of a pure lipid bilayer can be increased by a factor of 2. The inclusion of surfactant considerably reduced both the extensibility and the maximum stress that the bilayer could withstand. This may explain why dividing cells are more at risk than non-growing cells. The leakage studies showed that both C10E6 and C12E6 alcohol ethoxylates were capable of damaging the cytoplasmic membrane. However, there appear to be subtle differences in the mode of action of the two surfactants. C10E6 had very little effect below its MIC and as such may require an accumulation of surfactant at the site of action. A threshold concentration must then be reached for damage to occur. In contrast, C12E6 exerted its effects below its MIC, suggesting catastrophic injury and an ability to partition almost immediately into the cytoplasmic membrane. It is unlikely that these effects are due to the differing partition coefficients of the two surfactants, which span the optimum log P value of 4 for Gram-negative bacteria, since a similar amount of surfactant might be expected to be present in the cell membrane owing to the high propensity for the surfactants to partition from water. Therefore, it is proposed that these differences in interaction are due to the relative sizes of the hydrophobic tails of these compounds. Membrane phospholipids typically have an alkyl chain length of between 16 and 18. The longer tail group of C12E6 is more likely to insert itself into and flip-flop across the cytoplasmic membrane of the bacteria. Le Maire et al. [58] have demonstrated the ability of C12E8 to flip-flop across a variety of intact membranes within milliseconds and this is consistent with the constant partition of C12E6 into the bacterial cell. It is further proposed that, owing to the shorter chain length of C10E6, the head group will have a larger contribution to the hydrophilicity of the surfactant than that of C12E6. This is supported by the hydrophile–lipophile balance value of C10E6 (12.5) compared with that of C12E6 (11.8). The increased hydrophilicity of C10E6 may cause it to accumulate at the exterior of the membrane bilayer rather than flip-flop across, until the external leaf of the bilayer becomes saturated with surfactant. At this point, mixed phospholipid/surfactant micelles would

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form, resulting in complete transition [59]; this appears to occur at concentrations close to the MIC. At this stage, gross membrane damage occurs. The interaction of the surfactants with the membrane appears to occur when the surfactant is in monomeric form [60], as significant damage is caused to the bacterial cells before the CMC is reached for either surfactant.

[19]

[20]

[21]

Acknowledgment [22]

S.L.M. is grateful to Unilever Research Laboratories (Port Sunlight, UK) for funding this research project. [23]

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