Effect of Various Nonionic Surfactants on Growth

Transcription

1 JOURNAL OF BACTERIOLOGY, May, 1966 Vol. 91, No. 5 Copyright 1966 American Society for Microbiology Printed in U.S.A. Effect of Various Nonionic Surfactants on Growth of Escherichia colil MICHAEL J. ROSE, JR., STEPHEN A. ARON, AND BERNARD W. JANICKI Veterans Administration Hospital, Washington, D.C., and Department of Microbiology, University of Maryland, College Park, Maryland Received for publication 17 January 1966 ABSTRACT RosE, MICHAEL J., JR. (Veterans Administration Hospital, Washington, D.C.), STEPHEN A. ARON, AND BERNARD W. JANICKI. Effect of various nonionic surfactants on growth of Escherichia coli. J. Bacteriol. 91: Escherichia coli cultivated in media containing 0.5, 1.0, 2.0, or 4.0% concentrations of surfaceactive polyoxyethylene derivatives of formaldehyde polymers of octyl phenol (Triton WR-1339; Macrocyclon) or of sorbitan mono-fatty acid esters (Tween 20, 40, 60, and 80) exhibited significantly retarded growth only at the highest concentration. To determine the mechanism of bacteriostasis, certain derivatives and compounds related to the surfactants were investigated. Experiments with compounds related to the Triton-type agents demonstrated that incorporation of monomeric substances (Triton X-205, X-305, Igepal CA-730, or Dowfax 9N20) into the medium at a concentration of 4.0% did not inhibit the growth of E. coli. It was concluded that the formaldehyde polymer was essential for growth inhibition by the polyoxyethylene derivatives of octyl phenol. The inhibitory activity of the Tween compounds, in contrast, appeared to result from the unesterified fatty acids which contaminate the commercial preparations. Polyol (60), the sorbitan polyoxyethylene derivative of Tween 60 and the basic structural unit of all the Tween-type compounds, and a Tween 80 preparation which was purified by extraction of the unesterified oleic acid, were not inhibitory. Moreover, the amount of free oleic acid present as a contaminant of Tween 80 was found to be sufficient to cause significant growth inhibition. These results and the observation that E. coli does not appear to hydrolyze the esterified fatty acid of Tween 80 led to the conclusion that growth inhibition obtained with various Tween compounds probaby is a function of their respective fatty acid contaminants. Escherichia coli cultures grown in nutrient broth containing 0.5% Triton WR-1339, a nonionic surfactant, liberated fewer mature virulent phage particles on infection by the T2, T4, and T6 bacteriophages than did the control cultures (6; Janicki, Rose, and Aron, Proc. Intern. Congr. Surface Active Substances, 4th, in press). We also have observed that cultivation of the bacilli in increasing concentrations of this detergent resulted in a further reduction of burst size (Rose, Aron, and Janicki, Bacteriol. Proc., p. 17, 1965). Since burst size is partially a function of the physiology of the host cell, the possibility of a detrimental effect of the detergent on the bacilli alone could not be ignored. The purpose of this study was to examine the effect of incorporating various nonionic surfactants in the culture medium on the growth of E. coli. MATERIALS AND METHODS Two general types of nonionic surfactants were used. Polyoxyethylene ethers offormaldehyde polymers of octyl phenol. Triton WR-1339 was obtained from the Winthrop Laboratories (Rensselear, N.Y.), and I Presented in part at the 65th Annual Meeting of Macrocyclon was supplied by R. J. W. Rees of the the American Society for Microbiology, Atlantic National Institute for Medical Research (Mill Hill, City, N.J., April This material is part of a London, England). The latter was listed as compound thesis to be submitted by the senior author to the Department of Microbiology, University of Maryland, preparation by Cornforth and his co-workers (1). They no. HOC-20 (GN/57) in the report describing its in partial fulfillment of the requirements for the Ph.D. indicated that, although Triton appeared to be a degree. linear polymer and Macrocyclon was cyclic, both had 1863

2 1864 ROSE, ARON, AND JANICKI J. BACTERIOL. the same general constitution, being formaldehyde polymers of 4 octyl phenol units, each of which contained approximately 20 ethylene oxide groups in a chain linked to the phenolic nucleus by an ether bond. Compounds related to this type included Triton X-205, the monomer of Triton WR-1339 with a chain composed of 20 ethylene oxides, and Triton X-305, a monomeric octyl phenol with 30 ethylene oxides, which were supplied by Rohm & Haas Co. (Philadelphia, Pa.), as well as Igepal CA-730, a monomeric octyl phenol with 15 ethylene oxides, which was supplied by General Aniline and Film Co. (New York, N.Y.), and Dowfax 9N20, a monomeric nonyl phenol with 20 ethylene oxides, which was obtained from Dow Chemical Co. (Midland, Mich.). Polyoxyethylene ethers of sorbitan mono-fatty acid esters. Polyoxyethylene ethers of sorbitan esterified with lauric, palmitic, stearic, or oleic acid (Tweens 20, 40, 60, and 80, respectively) were obtained from Atlas Chemical Industries, Inc. (Wilmington, Del.). Polyol (60), the polyoxyethylene ether of sorbitan derived from Tween 60, was supplied by J. F. Treon of the Atlas Chemical Industries. In such compounds, a total of 20 ethylene oxide units are linked by ether bonds to the sorbitan mono-fatty acid esters in chains of varying length (7). Tween 80 was purified by extraction of unesterified oleic acid from the commercial product by use of the method outlined by Davis (3). Analysis of the extract by Davis' method (4) indicated that commercial Tween 80 contained approximately 0.6% unesterified oleic acid. After the free oleic acid had been extracted, the purified Tween 80 was dialyzed overnight against distilled water. On autoclaving, the purified material is converted to an insoluble gel in water; this feature was employed as a procedure for its concentration after dialysis. When cool, the water was decanted from the gel, and it was redissolved in sterile distilled water and stored at 5 C. Extraction of the unesterified oleic acid was found to be essentially complete (at least 95%) by incorporation of I'25-labeled oleic acid (Volk Radiochemical Co., Los Angeles, Calif.) into the Tween 80 specimen before extraction and subsequent analysis of the extract. Since prolonged storage, even at 5 C, results in a reappearance of free fatty acid (3), purified Tween 80 was used within 1 week of its preparation for all experiments. Purified Tween 80 labeled with I125 on the esterified oleic acid also was prepared. An 80-ml amount of a 12.5% Tween 80 solution in 0.9% NaCI was buffered to ph 8.6 with 20 ml of glycine-naoh buffer and was rapidly added to a flask containing 5 ml of a 0.2 M solution of ICI and 1 ml (50 juc) of NaI125 (Volk Radiochemical Co.). This mixture was then purified as above, with the exception that it was not autoclaved since this appeared to remove the label. An 8-g amount of the resultant purified Tween 80-I125 was dissolved in 60 ml of distilled water and dialyzed against distilled water overnight at 5 C. The dialyzed material was adjusted to 100 ml with distilled water and sterilized by filtration through a membrane filter (Gelman Polypore, Type AM 7). Labeling of the esterified oleic acid was confirmed by thin-layer chromatography of the radioactive extract from hydrolyzed purified Tween Hydrolysis was accomplished by refluxing the labeled purified Tween 80 for 1 hr in one-tenth its volume of concentrated HCI; the extract for chromatography was prepared by Davis' procedure (4). All of the above materials were prepared as sterile 8.0% solutions and stored at 5 C; with the exception of labeled purified Tween 80, sterilization was accomplished by autoclaving. Appropriate dilutions were made with sterile distilled water. Oleic acid (Fisher Scientific Co., Silver Spring, Md.) was prepared as an aqueous emulsion (0.1%) by use of a Branson Sonifier (model S-75), and was sterilized by autoclaving. Bacterial culture techniques. The B strain of E. coli (ATCC 11303) was used throughout the study. The influence of the various test materials on bacterial growth was determined by the standard plate count method; all plating was done in triplicate. Experimental and control media were prepared by diluting concentrated (twice) sterile nutrient broth (Difco) containing 10 g of NaCl per liter with an equal volume of sterile test solution or water. Cultures were prepared by inoculating 9.8 ml of medium with 0.2 ml of an overnight broth culture of E. coli; all cultures were prepared in quadruplicate, incubated at 37 C, and assayed for growth at selected intervals. In experiments designed to determine the ability of E. coli to hydrolyze the sorbitan-fatty acid ester linkage of the Tween-type compounds, broth containing 125-labeled purified Tween 80 at a final concentration of 4% and control broth were prepared. One-half of the test medium and an equal volume of the control medium were inoculated with an overnight culture of E. coli; the remainder of the test medium was used as an uninoculated control. All were incubated at 37 C for 3 hr, after which growth was assayed in the inoculated samples and the inoculated and uninoculated test media were passed through the membrane filter. A 5-mI portion of each filtrate was assayed for radioactivity and extracted by Davis' method to remove any unesterified labeled oleic acid which may have been liberated from the Tween 80 molecule. Extractable radioactivity was expressed as a percentage of the filtrate activity; the percentage of extractable radioactivity in the inoculated test medium was compared with that in the uninoculated test medium as a measure of bacterial hydrolysis of Tween 80. Radioactivity measurements. All radioactivity measurements were made with a differential scintillation spectrometer (model 530, Baird Atomic, Cambridge, Mass.) by use of either the Isomatic well sample changer (model 707) with a 3-inch well crystal or a bulk counter with a 5-inch well crystal. Statistical analysis. The statistical significance of the differences between mean values for growth in control and experimental cultures was analyzed by the methods outlined by Croxton (2). The differences were considered to be significant if the probability was less than 0.01.

3 VOL. 91, 1966 EFFECT OF SURFACTANTS ON E. COLI GROWTH 1865 RESULTS The results of population studies demonstrated that incorporation of the polyoxyethylene derivatives of either formaldehyde polymers of octyl phenol or soibitan mono-fatty acid esters into the growth medium at a final concentration of 0.5% had no inhibitory effect on the growth of E. coli (Fig. 1). The 3-hr culture was selected as a suitable sampling time for subsequent experiments, since it appeaied to mark the end of the logarithmic growth phase and served as the inoculum for our earlier studies (6). A series of experiments were perfoi med to determine the effect of various concentrations of the surfactants on growth of E. coli after 3 hr of incubation at 37 C. Pieliminary experiments were conducted with Triton WR-1339; the results of a typical experiment are represented in Fig. 2. Significant inhibition of growth was observed only with broth containing Triton at a final concentration of 4%. Twelve such experiments were performed; the mean per cent growth inhibition and its standard deviation were 30 and 10, respectively, for the 4% Triton cultures. Similar experiments were performed with the other surfactants; all agents tested significantly inhibited growth of E. coli at the 4% concentration. At this concentration, the Tween compounds appeared more inhibitory than either Triton or Macrocyclon (Fig. 3), but this difference was not significant. Additional experiments were designed to determine whether the surfactants were exerting a s Hours of Incubation at 37- C FIG. 1. Growth of Escherichia coli in nutrient broth and in broth containing various nonionic surfactants at afinal concentration of 0.5%. (The log per cent growth at each time interval was derived by expressing the viable-cell count of each culture as a percentage of its viable-cell count at zero-time.) o u - 40 ' % 0.5% 1.0% 2.0% 4.0% Concentration of Triton WR FIG. 2. Growth of Escherichia coli after 3 hr of incubation at 37 C in nutrient broth and in broth containing various concentrations of Triton WR (The per cent growth for each concentration was obtained by expressing the viable-cell count of the surfactant cultures as a percentage of the viable-cell count of the control culture.) 6-'l q , CONTROL TRITON MACRO- TWEEN TWEEN TWEEN TWEEN WR-1339 CYCLON FIG. 3. Growth of Escherichia coli after 3 hr of incubation at 37 C in nutrient broth and in broth containing various nonionic surfactants at a final concentration of4%. (The per cent growth was obtained by expressing the viable-cell count ofeach surfactant culture as a percentage of the viable-cell count of the control culture.) lethal or a static effect on the cultures. For this purpose, viable-cell counts were performed on 4.0% surfactant and control cultures immediately after inoculation and after 15 and 60 min of incubation. The results of such experiments are presented as a composite in Fig. 4. No significant reduction of viable cells was apparent with any of the agents, indicating that growth inhibition reflected a bacteriostatic action of the surfactants. As in Fig. 3, it appeared that the Tween

4 1866 ROSE, ARON, AkND JANICKI J. BACTERIOL. compounds behaved similarly as a group and were slightly more active than Triton or Macrocyclon. In an effort to describe the nature of the surfactant-induced bacteriostasis, experiments were performed in which compounds related to the surfactants were incorporated into the growth medium at a 4% concentration. The results of a typical experiment conducted with monomeric derivatives of Triton WR-1339 and Macrocyclon are shown in Table 1. The data demonstrated that growth of E. coli in media containing 4.0% Triton WR-1339 was significantly inhibited, but no inhibition occurred when the monomers were incorporated into the culture medium at a 4% concentration. Subsequent experiments were conducted with derivatives of the Tween compounds. Growth of E. coli in media containing e Control Triton WR El Macrocyclon El Tween 20 Tween 40 Tween 60 Tween 80 o 15 6 Minutes of Incubation at 37 c FIG. 4. Growth of Escherichia coli in nutrient broth and in broth containing various nonionic surfactants at a final concentration of4%. (The per cent growth at each time interval was obtained by expressing the viable-cell count of each culture as a percentage of its viable-cell count at zero-time.) TABLE 1. Effect of 4.0% concentrations of Triton WR-1339 or various derivatives on growth of Escherichia coli after 3 hr of incubation at 37 C Culture Viable-cell count (X 106/ml) Triton Control Igepal Dowfax WR- ~~~CA-730 9N20 WR-9 X-205 X-305 4% Polyol (60) was not inhibited while significant inhibition was found with the culture containing 4% Tween 60, the parent compound (Table 2). Similar experiments were performed with Tween 80 and with a Tween 80 preparation which was purified by removal of unesterified oleic acid; each was incorporated into the culture medium at a 4% concentration. The results of a typical experiment are presented in Table 3. It was obvious that removal of the unesterified oleic acid contaminant from Tween 80 resulted in a loss of its inhibitory ability. The results also show that growth was inhibited when oleic acid was incorporated into the medium at a concentration of 0.025%; this amount of oleic acid was calculated to be present in 4% Tween 80 nutrient broth from the data of extraction experiments. In a final series of experiments, an attempt was made to determine whether the esterified fatty acid of the Tween-type agents is subjected to enzymatic hydrolysis by E. coli. For this purpose, a purified Tween 80 preparation which was labeled with F125 on its esterified oleic acid was added to the culture medium at a 4% concentration. One-half of the medium was inoculated and the other was not; viable-cell counts were performed on the inoculated cultures and on nutrient broth control cultures after incubation at 37 C for 3 hr. After incubation, filtrates of the inoculated and uninoculated media containing the labeled purified Tween 80 were extracted to remove any free oleic acid, and the radioactivity of the extracts was measured. The results of a typical experiment which are shown in Table 4 demonstrated that the labeled purified Tween 80 did not inhibit growth. The data also indicated that no bacterial hydrolysis of the esterified fatty acid occurred during this interval of incubation, since the amount of extractable radioactivity of the inoculated media was essentially the same as that of the uninoculated media. TABLE 2. Effect of 4% concentrations of Tween 60 or Polyol (60) on growth of Escherichia coli after 3 hr of incubation at 37 C Expt Per cent of control growth Tween 60 Polyol (60) Mean Mean 38 94

5 VOL. 91, 1966 EFFECT OF SURFACTANTS ON E. COLI GROWTH 1867 TABLE 3. Effect of 4% concentrations of Tween 80 or purified Tweeni 80 or 0.025% concentration of oleic acid on growth of Escherichia coli after 3 hr of incubation at 37 C Viable-cell count (X 106/ml) Culture - Control Tween 80 Tween 80 Oleic Mean TABLE 4. Growth of Escherichia coli and extractable radioactivity in nutrient broth containing I125-labeled purified Tween 80 at a 4% concentration Culture Viable-cell count Per cent extractable (X 106/ml) radioactivity Control 1125-Tween Uninocu- Inoculated 80 lated Mean DisCussIoN In the present investigation, the observation that incorporation of Triton WR-1339 at a 0.5% concentration in the growth medium of E. coli had no inhibitory effect on the growth of the bacilli (6) was extended to include surfactants representing two chemical types, the polyoxyethylene ethers of formaldehyde polymers of octyl phenol and of sorbitan-mono fatty acid esters (Fig. 1). The results of this study also suggested that the progressive decrease in burst size previously observed with increasing concentrations of Triton WR-1339 (Rose et al., Bacteriol. Proc., p. 17, 1965) may be explained, at least partially, by the inhibition of growth caused by higher concentrations of this surfactant (Fig. 2). A similar inhibition of growth was observed when Macrocyclon, Tween 20, 40, 60, or 80 was incorporated into the culture medium at a concentration of 4% (Fig. 3); all surfactants exhibited bacteriostasis at this concentration (Fig. 4). Although both general types of surfactants were significantly bacteriostatic, the mechanism of the bacteriostatic effect of the Triton-type agents appears to differ from that of the Tween compounds. Since the monomeric derivatives of Triton and related compounds were not inhibitory (Table 1), it seems reasonable to believe that growth inhibition by agents of this type may be a function of their polymeric nature. It has been suggested that Triton and Macrocyclon inhibit the action of lipolytic enzymes by enclosing the substrate within their folded structure (Jorolan and Janicki, Biochem. Pharmacol., in press). A similar inhibition of E. coli enzymatic activity can be suggested as a tentative explanation for growth inhibition with such agents; the glucose dehydrogenase activity of E. coli cultivated in nutrient broth containing Triton at a final concentration of 0.5% was significantly less than that of control cultures (Janicki et al., in press). The inhibitory activity of the Tween compounds, in contrast, appears to be a direct consequence of the unesterified fatty acids which contaminate the commercial preparations. Polyol (60), the sorbitan polyoxyethylene ether of Tween 60 and the basic structural unit of all the Tween-type compounds, did not inhibit growth of E. coli (Table 2). This observation and the fact that purification of Tween 80 by extraction of the unesterified fatty acid resulted in a noninhibitory preparation (Table 3) strongly implicated the fatty acid as the inhibitory material. The amount of free oleic acid present as a contaminant of Tween 80 was found to be sufficient to cause a significant inhibition of growth, although not to the same degree as Tween 80 (Table 3). It should be mentioned that the physical state of the fatty acid in these cultures may have been responsible for this difference in inhibitory activity. In the oleic acid-nutrient broth preparation, an aqueous emulsion of oleic acid was incorporated in nutrient broth, whereas in Tween 80-nutrient broth the oleic acid contaminant may have been solubilized under the influence of the surfactant. On this basis, even though equivalent amounts of fatty acid were present in both preparations, it appears reasonable to believe that Tween 80 broth may have contained oleic acid in a more active state. It also is of interest that the growth of tubercle bacilli is similarly inhibited by the unesterified fatty acid of Tween 80 and that cultivation of the tubercle bacillus in the Tween compounds is further complicated by its ability to hydrolyze the esterified fatty acids (5). However, we found no evidence to indicate that growth inhibition of E. coli involves hydrolysis of the Tween molecule. Using

6 1868 ROSE, ARON, AND JANICKI J. BACTERIOL. In'-labeled purified Tween 80, we observed no significant bacterial hydrolysis during the period of bacteriostasis (Table 4). Thus, on the basis that the polyol moiety of the Tweens is not inhibitory and that E. coli does not hydrolyze the Tweens, it appears valid to suggest that the growth inhibition obtained with the various Tween compounds is a function of their respective fatty acid contaminants. ACKNOWLEDGMENTS The assistance in preparing the labeled material provided by A. Miale of the Radioisotope Service of the Veterans Administration Hospital, Washington, D.C., and S. Lakshmanan of the Department of Chemistry at the University of Maryland is gratefully acknowledged. LITERATURE CITED 1. CORNFORTH, J. W., P. D'A. HART, G. A. NICHOLLS, R. J. W. REES, AND J. A. STOCK Antituberculous effects of certain surface-active polyoxethylene ethers. Brit. J. Pharmacol. Chemotherap. 10: CROXTON, F. E Elementary statistics with applications in medicine and the biological sciences. Dover Publications, Inc., New York. 3. DAVIS, B. D The preparation and stability of fatty acid-free polyoxyethylene sorbitan monooleate ("Tween" 80). Arch. Biochem. 15: DAVis, B. D The estimation of small amounts of fatty acid in the presence of polyoxethylene sorbitan partial fatty acid esters ("Tween") and of serum proteins. Arch. Biochem. 15: DAVIS, B. D., AND R. J. DUBOS Interaction of serum albumin, free and esterified oleic acid and lipase in relation to cultivation of the tubercle bacillus. Arch. Biochem. 11: ROSE, M. J., JR., S. A. ARON, AND B. W. JANICKI Influence of nonionic surfactants on bacteriophage infections. I. Effect of Triton WR on T2 coliphage infection. J. Bacteriol. 87: TREON, J. F Physiological properties of selected nonionic surfactants. Proc. Sci. Sect. Toilet Goods Assoc. 40: Downloaded from on October 13, 2018 by guest