Effects of a Quaternary Ammonium Compound on Escherichia colil

CEGLOWrSKI, W. S. (New Jersey Agricultural Experimental Station, New Brunswick, N. J.) AND S. A. LEAR. Effects of a quaternary ammonium compound on Escherichia coli. Appl. Microbiol. 10:458-462. 1962-Increasing amounts of tetradecyldimethylbenzyl ammonium chloride (TAC) were lethal to an increasing proportion of an actively growing culture of Escherichia coli. The loss of nucleic acid material by actively growing E. coli did not appear to play a major role in the lethal effect. It was found that lag-phase cells were more sensitive than logarithmic-phase cells to the lethal effect of TAC. The effect of TAC on the lysozyme sensitivity of the test organism was compared with that obtained using disodium dihydrogen ethylenediaminetetraacetate (EDTA). Although TAC was found to render the test organism susceptible to lysozyme, the degree of lysis never reached that attained with EDTA. In the dairy and food industries, the quaternary ammonium compounds have found wide use as sanitizing agents. In their practical application, these compounds may be used in cases where bacteria are actively growing. In this study, we investigated the efficacy and some of the effects of a quaternary ammonium compound on Escherichia coli. MATERIALS AND MIETHODS Growth experiments. The test organism was E. coli ATCC 11229. From a supply of ampoules of lyophilized culture prepared at the start of the study, one ampoule was used to inoculate a flask of nutrient broth. After a suitable period of incubation, this broth was used to inoculate a supply of nutrient agar slants. After 3 hr of incubation at 37 C, the slants were stored at 5 C for no longer than 6 weeks. A new ampoule was opened whenever a fresh supply of inoculated slants was required. In preparation for an experiment, a nutrient agar slant of the organism was transfered by pipette to a portion of the medium of the composition given by Fisher and I Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, The State University, Department of Dairy Science, New Brunswick, N. J. Portion of a dissertation presented by the senior author as partial fulfillment of the requirements for the Ph.D. degree. 2 Present address: Plum Island Animal Disease Laboratory, Greenport, Long Island, N. Y. Armstrong (1947), using glucose as a carbon source. The inoculated flask was incubated on a shaking water bath (New Brunswick Scientific Company, New Brunswick, N. J.) for 4 to 6 hr. In another flask of medium, a subculture was made and incubated for 8 to 10 hr. Another subculture was made in either 500 ml or 1 liter of medium, which was incubated for 3 to 4 hr on a reciprocating shaker (Arthur H. Thomas Co., Philadelphia, Pa.). At the end of this growth period, the culture was chilled in an ice bath, harvested by centrifugation, and washed twice with saline. The cells were then resuspended in fresh medium to a predetermined turbidity. Quantities (100 ml) of the culture were then transferred to 300-ml flasks. A 1-ml amount of an appropriate concentration of the quaternary ammonium compound was added to each experimental flask. The flasks were then incubated in a shaking water bath at 37 C and observed over a period of 3 to 6 hr. The quaternary ammonium compound used in this study was tetradecyldimethylbenzyl ammonium chloride (TAC). TAC was kindly supplied by the Rohm and Haas Company, Philadelphia, Pa. A stock solution of TAC was prepared and stored at 5 C. For the experiments, the stock solution was diluted in a volumetric flask, so that the desired amount of TAC was contained in 1 ml of solution. Preliminary growth experiments were observed over a 6-hr period. These cultures were examined by following turbidity changes, which were judged by observing the absorbancy of the cultures at wavelengths of 260 and 420 m,u in a Beckman DU spectrophotometer (Beckman Instruments Inc., Fullerton, Calif.), according to the method of Wisseman et al. (1954). Other experiments were performed and observed in more detail over a period of 3 hr. At predetermined time intervals, samples of culture were removed from the flasks, and the turbidity, viable plate count, and the ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein content of the cultures was determined. The viable plate count was determined by serial dilution of a sample of culture after inactivation of the TAC with the diluent of Weber and Black (1948). Samples (1 ml) of suitable dilutions were plated in duplicate on eosin methylene blue and nutrient agar. The plates were incubated for 36 to 48 hr at 37 C and then counted. Samples of culture were centrifuged in a Servall SS-1 angle-head centrifuge (Ivan Sorvall, Inc., Norwalk, Conn.) and the cells were fractionated into an RNA, DNA, and protein fraction by the procedure of Richmond (1959). Ribose was determined by the method of Mejbaum 458 on A uust 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom EFFECT OF AMMONIUM COMPOUNDS ON E. COLI (1939). To express the results in terms of RNA, the ribose equivalents of a sample of purified yeast RNA (kindly supplied by F. F. Davis, Rutgers, The State University, New Brunswick, N. J.) was determined. DNA was determined spectrophotometrically, using a sample of highly polymerized DNA (Nutritional Biochemical Corp., Cleveland, Ohio) as the standard. Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin (Armour Laboratories, Kankakee, Ill.) as the standard. The supernatant culture fluids were made to contain 0.2 N perchloric acid, and the absorption values at 5-m, intervals from 240 to 280 m,u were determined. The supernatant fluids were also lyophilized, reconstituted in distilled water or pyridine, and chromatographed using the system of Wyatt (1951). Another series of experiments was performed to determine the influence of the physiological state of the inoculum on the resistance of the test organism to TAC. The cells used as the inoculum in these experiments were grown in the glucose medium, harvested by centrifugation, washed twice with saline, resuspended in distilled water, and held at 5 C for 18 hr. Other experiments were performed to determine the effect of growth in a glucose medium containing TAC on re-exposure to TAC. In these experiments, cells which had been grown in a glucose medium containing TAC were harvested, washed twice with saline, and resuspended in a fresh medium containing TAC. Lysozyme study. The effect of TAC onthe susceptibility of E. coli to the action of lysozyme was studied, using the system of Repaske (1958). The test organism was grown in glucose medium, harvested by centrifugation while in the early logarithmic phase of growth, and washed twice with saline. Experimental observations were made in silica cells containing 3 ml of solution with the following composition: tris(hydroxymethyl) aminomethane buffer (pH 8.0), 100 ,M; lysozyme (California Corporation for Biochemical Research, Los Angeles, Calif.), 100 Mg; disodium dihydrogen ethylenediaminetetraacetate (EDTA; Hach Chemical Co., Ames, Iowa), 400 Mug; and sufficient cell suspension to give an A660 of 0.500. Various amounts of TAC were substituted for the chelating agent and lysis was followed by measuring the change in the A660 with time. RESULTS AND DISCUSSION The results of the preliminary growth experiments are presented in Fig. 1. For convenience, the 0 time A420 reading of the control culture, which was adjusted to 0.100, was assigned a value of 1.00; all the other readings are expressed as ratios of this value. The growth curves demonstrate that concentrations of TAC from 5 to 10 Mug per ml will inhibit the growth of E. coli. Over a 6-hr observation period, the A420 of the control culture increased almost 13-fold. Increasing amounts of TAC resulted in progressively diminished turbidity increases during the 6-hr period. Finally, at TAC concentrations of 8 and 10 MAg per ml, the turbidity of the cultures decreased upon incubation. The term bacteriostatic has been used frequently in reports concerning bactericides, where only end-point methods have been used to determine the effectiveness of a bactericide. Under these conditions, the term usually means that, when exposing a particular starting concentration of bacteria to a certain level of bactericide for a given period of time, surviving cells can be isolated. Originally it was thought that a TAC level might be found which would inhibit growth and yet result in only a slight lethal effect over the experimental period. If an end-point method had been used in the present study, it would appear that only bacteriostatic levels of TAC were used. Actually, all levels of TAC utilized were bactericidal, since it will be shown that significant reductions in the numbers of viable cells took place during the experimental period. The experiments were observed in greater detail for a 3-hr observation period (Table 1). The results indicate that a given proportion of the cells in the inoculum are killed within a relatively short period of time, and the remainder of the population continues to grow. Increasing amounts of TAC are apparently lethal to an increasing proportion of the population. In the cultures which exhibited growth, increases in RNA, DNA, and protein were noted. If pronounced lysis is the primary effect of TAC, one might expect large reductions in the intracellular nucleic acid content of the exposed cultures with the appearance of nucleic acid materials in the supernatant culture fluids. The absorbancy values of the culture supernatant fluids at 0 and 3 hr are presented in Table 2. It should be noted that at least 30 min elapsed before the cells were removed by centrifugation. The absorbancies of glucose medium containing 5 and 10 Mg of TAC per ml are included for