Kinetic Analysis of the Bactericidal Action of a Bis-quaternary Ammonium Compound against Escherichia coil

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1 Biocontrol Science, 2003, Vol.8, No.4, Original Kinetic Analysis of the Bactericidal Action of a Bis-quaternary Ammonium Compound against Escherichia coil TOMOKO SUMITOMO, TAKUYA MAEDA, HIDEAKI NAGAMUNE, AND HIROKI KOURAI* Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima, Minamijosaniirna-cho, Tokushima , Japan Received 13 December 2002/Accepted 24 June 2003 The bactericidal activity of bis-quaternary ammonium compounds (bis-qacs) such as 4,4'- (1,6-hexamethylenedithio)bis(1-octylpyridinium bromide) (4DTBP-6,8), which possess two quaternary ammonium cations in the molecule, is stronger than that of mono-qac possessing one ammonium cation in the molecule. It is also reported that the bactericidal action of bis- QACs is less influenced by the molecular hydrophobicity of the drug or environmental conditions such as temperature or ph than that of mono-qac. In order to clarify the mode of bactericidal action of a bis-qac, 4DTBP-6,8, against Escherichia coli, its bactericidal activity was kinetically elucidated compared with that of a mono-qac, N-octylpyridinium bromide (P- 8). Though there was little difference in the dilution coefficient (n) of 4DTBP-6,8 and P-8, the minimum effective concentration for the bactericidal action of 4DTBP-6,8 was different from that of P-8. The rate constant (k) of the bactericidal action of 4DTBP-6,8 was little influenced by the treatment temperature and the growth temperatures, and the Arrhenius plots of the k values showed a linearity. The apparent activation energy (Ea) for the bactericidal action of 4DTBP-6,8 was 15.6kJ/mol. On the other hand, the k values of P-8 were extremely influenced by the treatment and growth temperatures, and the Arrhenius plots of the k values gave a broken line. The calculated E. value was 99.8kJ/mol at a low temperature (10-30 Ž) and 40.6kJ /mol at a high temperature (30-40 Ž) Judging from these findings, it is clear that the mechanism of bactericidal action of bis-qac is different from that of mono-qac. Key words : Quaternary ammonium compound/bactericidal action/rate constant/arrhenius plots/ Activation energy. INTRODUCTION Quaternary ammonium compounds (QACs) have been widely used as disinfectants in the food industry and hospitals, because they have comparatively low toxicity and affect a wide range of microbes. Various kinds of QACs have been synthesized and characterized, and their bactericidal mechanism has been studied (Baley et al., 1977; Devinsky et al., 1991; Kourai et al., 1980 and 1994a; Krysinski, 1990 and 1991; Maeda et al., 1996; Okazaki et al., 1996, 1997a and *Corresponding author. Tel : , Fax : b). It was shown that those disinfectants are adsorbed on the bacterial cell surface, then invade and destroy the cell wall and the cell membrane. However, those studies have been performed using mono- QACs with one ammonium cation in their molecules. Recently, we reported the synthesis and the antimicrobial characteristics of bis-qacs possessing two quaternary ammonium atoms in their molecules (Maeda et al., 1998 and 1999; Okazaki et al., 1997c; Yoshida et al., 2000). The bactericidal activity of bis- QACs was shown not to be affected by the length of their hydrophobic alkyl chain, unlike that of mono- QACs. Furthermore, the activity of the bis-qacs was comparatively uninfluenced by environmental

2 146 T. SUMITOMO ET AL. conditions such as ph or temperature. It was implied that the excellent properties of bis-qacs are derived from their dimeric structure. It was suggested that bis- QAC might have a bactericidal mechanism different from that of mono-qacs. Studies on the bactericidal action of disinfectants have been performed so far by monitoring their minimum bactericidal concentration (MBC). However, it is difficult to clarify the point at which disinfectants attack bacteria by using the MBC value as an index, because the drug at the MBC dose completely kills the bacteria. In order to clarify the bactericidal mechanism of QACs, it is important to examine the ratelimiting step in the bactericidal action of QAC. We now focus on the kinetics of the bactericidal actions of bis- QAC and mono-qac. In this study, as one of the indices which clarify the mode of the bactericidal action of QACs against Escherichia coli, the kinetics of the bactericidal action of bis-qac were examined and compared with those of mono-qac.. MATERIALS AND METHODS Microbe and preparation of cell suspension Escherichia coli IFO was used for all experiments. E. coli was preincubated in Luria-Bertani broth consisting of 1% (w/v) tryptone (OXOID, Hampshire, England), 0.5% (w v) yeast extract (0X01 D, Hampshire, England), and 0.5% (w/v) sodium chloride (Kanto Chemicals, Tokyo) for 18h at 37 Ž. The cell suspension (5m1) inoculated into 100 ml of nutrient broth (NB, BECTON DICKINSON, Sparks, MD, USA) was incubated until the middle exponential phase was reached (OD ) at the temperature of 20, 30 or 37 Ž. The grown cells were harvested aseptically by centrifugation at 5000 x g for 10min at 4 Ž and washed twice with sterile ice-cooled distilled water, and the cells were suspended and adjusted to a concentration of 2 x 106 cells/ml with sterile ice-cooled distilled water. Quaternary ammonium salts The chemical structures of 4,4'- (1,6-hexamethylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) and N-octylpyridinium bromide (P-8) are shown in Fig. 1. The synthesis procedures, their chemical properties and purities were previously described (Okazaki et al., 1997c). Measurement of bactericidal activity 4DTBP-6,8 solutions (0.1, 0.5, 0.7, 1.0, 1.5, 3.0 or 5.0ppm) and P-8 solutions (100, 500, 10000, 2000, 2500, 5000 or 10000ppm) were prepared in sterile distilled water. The cell suspensions (2 x 106 cells/ FIG. 1. Chemical structures of 4,4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium bromide) (4DTBP-6,8) and N-octylpyridinium bromide (P-8). ml) and antimicrobial solutions were each incubated in a water bath shaker for 5 min at various temperatures (10-40 Ž). The cell suspension (5 ml) was then poured into a 200-ml flask containing the antimicrobial solution (5 ml), and incubated for various periods (0-180 min) at various temperatures (10-40 Ž ). Immediately thereafter, aliquots (0.5 ml) of the mixtures were taken out and diluted serially with 4.5 ml of SCDLP medium (Wako Pure Chemicals, Osaka), followed by spreading the dilution mixtures on SCDLP agar plates (Wako Pure Chemicals, Osaka). After incubation at 37 Ž for 24h, the number of viable cells in the sample was counted. As a control, the number of viable cells in an untreated cell suspension was estimated. Kinetic analysis The tangent in the initial stage of bactericidal reaction of QACs is supposed to a first-order reaction in equation (1), dn/dt = kn (1) where N is the number of viable cells per unit volume at contact time t (min), and k (min-1) is the rate constant for bactericidal action. The k value is affected by the concentration of QACs, as expressed by the equation (2), k = acn (2) where n is the dilution coefficient and a is a constant. For the temperature dependency of k, the Arrhenius equation in the equation (3) was introduced. k = A e-ea/" (3) Where A is the frequency factor, Ea is the activation energy of bactericidal action, R is the gas constant, and T is the treatment temperature (K). RESULTS AND DISCUSSION

3 KINETIC ANALYSIS OF BACTERICIDAL ACTION OF BIS-QAC 147 Bactericidal activity The bactericidal activities of 4DTBP-6,8 and P-8 against Ecoli measured at 30 Ž are shown in Fig. 2. 4DTBP-6,8 did not strongly reduce the number of viable cells at 0.1 ppm, but the activity was extremely increased with the rise in the drug concentration. The survival curve indicating treatment with P-8 for the first 10min was similar to that with 4DTBP-6,8, but the activities of P-8 were extremely increased after 30min. Treatment of cells with sterile distilled water caused no reduction in viability (data not shown). Using equation (1) and (2), the effects of the concentration of 4DTBP-6,8 or P-8 were analyzed (Fig. 3). n values of 4DTBP-6,8 and P-8 were 1.22 and 1.10, respectively. From the intercept of the line with log k value (-2.16) in the absence of QACs, the minimum effective concentration for the bactericidal ac- FIG.3. Effect of the concentration of 4DTBP-6,8 or P-8 on the rate constant for the bactericidal action. Symbols: œ 4DTBP-6,8; Z, P-8; absence of QACs. Bars stand for the standard deviation of the mean. tion of 4DTBP-6,8 and P-8 were 0.045ppm and 121ppm, respectively. This implied that the bactericidal activity of 4DTBP-6,8 is about 2500 times higher than that of P-8. Temperature dependency of the bactericidal activity In order to compare the kinetics of the initial stage of bactericidal action of 4DTBP-6,8 with that of P-8, 4DTBP-6,8 (1ppm) and P-8 (2000ppm), at which the bactericidal abilities of Ecoli for 30min were equal to each other, were used in the following experiments. The increased treatment temperature tends to in- crease the bactericidal activities of QACs (Kourai et al., 1994a and 1994b). The bactericidal activities of 4DTBP-6,8 and P-8 were measured at various temperatures (10, 15, 20, 25, 30, 35 and 40 Ž). The survival curves of cells treated with 4DTBP-6,8 (1ppm) and P-8 (2000ppm) were affected by the treatment temperature as shown in Fig. 4. The curves of 4DTBP- 6,8 and P-8 showed that the activities increased with the increasing temperatures from 10 to 40 Ž. In order to examine the rate-limiting step of the bactericidal action of 4DTBP-6,8 and P-8, for all the survival curves of cells treated with 4DTBP-6,8 or P-8 at various temperatures, the rate constants for the bactericidal action, k, were evaluated from the slopes of the initial stage (1 min) of the survival curves. At the tempera- FIG. 2. Survival curves of Ecoli cells treated with 4DTBP-6,8 or P-8 at 30 Ž. Symbols: Z, 0.1ppm or 100ppm ; œ, 0.5ppm or 500ppm ;, 0.7ppm or 1000ppm ;, 1.0ppm or 2000ppm ; 1.5ppm or 2500ppm ; Ÿ 3.0ppm or 5000ppm ;, 5.0ppm or 10000ppm. Bars stand for the standard deviation of the mean. tures from 10 to 40 Ž, the k values of 4DTBP-6,8 and P-8 increased from 1.44 to 2.77min-1 and to 3.19min-1, respectively. The Arrhenius plots of the k values for the bactericidal action of 4DTBP-6,8 and P- 8 are shown in Fig. 5. The k values of P-8 were influenced by the treatment temperature, and the plots gave a broken line. On the other hand, the k values of

4 148 T. SUMITOMO ET AL. FIG.5. Arrhenius plots of the rate constant for the bactericidal action of 4DTBP-6,8 (1ppm)and P-8 (2000ppm). Symbols: œ, 4DTBP-6,8; Z, P-8. Bars stand for the standard deviation of the mean. -40 Ž) and E2 (10-30 Ž) were 40.6kJ / mol and 99.8kJ/mol, respectively. Such a difference between El (at high temperatures) and E2 (at low temperatures) may suggest that the interaction between P-8 and the bacterial cell membrane might be affected by the treatment temperature. Effect of the growth temperature of cells on bactericidal activity FIG.4. Effect of treatment temperature on the bactericidal activity of 4DTBP-6,8 (1 ppm) or P-8 (2000ppm). Symbols: Z, 10 Ž; œ, 15 Ž;, 20 Ž;, 25 Ž; ž, 30 Ž; Ÿ, 35 Ž;, 40 Ž. Bars stand for the standard deviation of the mean. 4DTBP-6,8 were little influenced by the treatment temperature in comparison with P-8. These results suggested that the bactericidal action of 4DTBP-6,8 in the initial stage is the simple Arrhenius type. Activation energy of the bactericidal action Two slopes in the Arrhenius plots for P-8 were steeper than the slope for 4DTBP-6,8 (Fig. 5). The Ea shows the chemical potential of the rate-limiting step in the bactericidal action calculated in the equations (3) using the data shown in Arrhenius plots. The Ea calculated from the slope of the Arrhenius plots of 4DTBP-6,8 was 15.6kJ/mol. The rate-limiting step in the bactericidal action of 4DTBP-6,8 was suggested to be an electrical adsorption and/or an ionic reaction of the drug on the anionic site of the bacterial surface. On the other hand, the calculated Ea values were different according to the temperature range, i.e., El (30 Decreasing the growth temperature causes an increase in the proportion of unsaturated fatty acids in cell membrane phospholipids of Ecoli (Marr et al., 1962). We compared the k values in the initial stage of the bactericidal action of 4DTBP-6,8 and P-8 at various treatment temperatures (10, 15, 20, 25, 30, 35 and 40 Ž), using cells grown at 20, 30 and 37 Ž in relation to the difference in their fatty acid compositions. The Arrhenius plots of k for the bactericidal action of 4DTBP-6,8 and P-8, for 20 Ž-, 30 Ž- and 37 -growth cells, are shown in Fig. 6. The k values of Ž P- 8 were influenced by the treatment temperature, and the plots gave broken lines. The temperature of the intersection shifted to the high temperature region with an increase in the growth temperature. On the other hand, the plots of 4DTBP-6,8 showed linearity independent of the growth temperature. It was suggested that the bactericidal action of 4DTBP-6,8 was not influenced by the composition of membrane lipids, unlike that of P-8. Judging from these findings, it is suggested that the bactericidal action of bis-qac is concerned with a different mechanism from that of mono-qac. In addition, it is suggested that the rate-limiting step in the bactericidal action of bis-qac might be the electrical

5 KINETIC ANALYSIS OF BACTERICIDAL ACTION OF BIS-QAC 149 FIG. 6. Effect of the growth temperature of cells on Arrhenius plots of the rate constant for the bactericidal action of 4DTBP-6,8 (1ppm) and P-8 (2000ppm) against Ecoli grown at 20 Ž ( œ), 30 Ž ( Z) and 37 Ž ( Ÿ). Bars stand for the standard deviation of the mean. adsorption on the bacterial surface; in contrast, that of mono-qac is closely related to the nature of the bacterial cell membrane. However, to clarify the detailed mechanism of the adsorption step of bis-qac, further investigation is now in progress. REFERENCES Baley, G.J., Peck, G.E., and Banker, G.S. (1977) Bactericidal properties of quaternary ammonium compounds in dispersed systems. J. Pharm. Sci., 66, Devinsky, F., Masarova, L., Lacko, I., and Mlynarcik, D. (1991) Structure-activity relationships of "soft" quaternary ammonium amphiphiles. J.Biopharm. Sci., 2, Kourai, H., Horie, T., Takechi, K., and Shibasaki, I. (1980) The antimicrobial characteristics of quaternary ammonium salts and their alkyl chain length (in Japanese). Bokin Bobai, 8, Kourai, H., Hasegawa, Y., Goto, S., and Nakagawa, K. (1994) Bacterioclastic action of dodecylpiridinium iodide against Escherichia coli K12 W3110. J.Antibact. Antifung. Agents, 22, Kourai, H., Oda, K., Takechi, H., and Nakagawa, K. (1994) The antimicrobial characteristics of poly [dimethyliminio (polymethylene) chloride] s. J. Antibact. Antifung. Agents, 22, Krysinski, J. (1990) Rough sets approach to analysis of the structure-activity relationship of quaternary imidazolium compounds. Arzneim. -Forsch., 40, Krysinski, J. (1991) Rough sets theory in structure-activity relationship analysis of quaternary pyridinium cornpounds. Pharmazie, 46, Maeda, T., Goto, S., Manabe, Y., Okazaki, K., Nagamune, H., and Kourai, H. (1996) Bactericidal action of N- alkylcyanopyridinium bromides against Escherichia coli K12 W3110. Biocontrol Sci., 1, Maeda, T., Okazaki, K., Nagamune, H., Manabe, Y., and Kourai, H. (1998) Bactericidal action of 4,4'-( a, co -poly methylenedithio) bis (1-alkylpyridinium iodide) s. Biol. Pharm. Bull., 21, Maeda, T., Manabe, Y., Yamamoto, M., Yoshida, M., Okazaki, K., Nagamune, H., and Kourai, H. (1999) Synthesis and antibacterial characteristics of novel biocides, 4,4'- (1,6-hexamethylenedioxydicarbonyl) bis (1-alkylpyridinium iodide) s. Chem. Pharm. Bull., 47, Marr, A. G. and Ingraham, J. L. (1962) Effect of temperature on composition of fatty acids of Escherichia coll. J. Bacteriol., 84, Okazaki, K., Maeda, T., Nagamune, H., and Kourai, H. (1997a) Synthesis and antibacterial characteristics of N- alkyl-4-alkylthiopyridinium bromides. Biocontrol Sci., 2, Okazaki, K., Maeda, T., Nagamune, H., and Kourai, H. (1997b) Antibacterial characteristics of N-alkyl-2 alkylthiopyridinium and N-alkyl-4-alkylthiopyridinium salts. Jpn. J. Toxicol. Environ. Health., 43, Okazaki, K., Maeda, T., Nagamune, H., Manabe, Y., and Kourai, H. (1997c) Synthesis and antibacterial characteristics of 4,4'- ( a, -polymethylenedithio) bis (1-alkylpyridi nium iodide) s. Chem. Pharm. Bull., 45, Yoshida, M., Maeda, T., Okazaki, K., Nagamune, H., Kunikata, K., Tsuchiya, H., Namba, T., and Kourai, H. (2000) Synthesis and antimicrobial characteristics of /V,AP-hexamethylenebis (4-carbamoyl-1-decylpyridinium bromide). Biocontrol Sci., 5,