Anti-Pasteurella pestis Factor

Transcription

1 JOURNAL OF BACTERIOLOGY, May 1968, p Vo1. 95, No. 5 Copyright 1968 American Society for Microbiology Printed in U.S.A. Anti-Pasteurella pestis Factor III. Effects of Fatty Acids on Pasteurella pestis D. M. EISLER AND E. K. VON METZ Naval Biological Laboratory, School of Public Health, Uniiversity of California, Berkeley, Califortnia Received for publication 11 March 1968 The bactericidal activity and the chemical and physical characteristics of lipid fractions of extracts from organs of normal mice and guinea pigs have been described previously. The present study describes the effects of commercially prepared fatty acids which are representative of those found in the extracts. Saturated fatty acids with 12, 14, and 16 carbon atoms and unsaturated fatty acids with more than one double bond were the most effective in killing Pasteurella pestis. Tweens 2 and 8, Spans 4 and 8, and some of the methyl esters of the fatty acids did not strongly inhibit P. pestis. It was concluded, therefore, that both the length of the carbon chain and the presence of the carboxyl group of the free fatty acids were important in their effects on P. pestis. Neither the very active lauric acid nor the relatively inactive oleic acid appeared to severely alter the morphology of P. pestis. Supernatant fluids from incubated mixtures of sodium laurate or sodium oleate and cells did not contain significantly greater concentrations of proteins or nucleic acids than did the controls. These observations do not preclude an alteration of the permeability of the cell walls. An anti-pasteurella pestis factor (APF) prepared from the organs of normal animals (3) proved to be a lipoprotein (2). Its antibacterial activity in vitro was ascribed to the lipid fraction which was held in solution by the protein. The lipid was separable by extraction with chloroform, and the component fatty acids of this antibacterial fraction were identified. The present report describes the antibacterial activities of commercial fatty acids representing those found in greatest concentrations in APF from mouse liver. MATERIALS AND METHODS Fatty acids and derivatives. Caproic, caprylic, lauric, myristic, palmitic, stearic, and linoleic acids were obtained from Matheson, Coleman, and Bell, Norwood, Ohio. Oleic acid was obtained from Braun-Knecht-Heiman Co., San Francisco, Calif. Linolenic acid and the methyl esters of lauric, myristic, palmitic, and linoleic acids were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. Tween 2 (polyoxyethylene sorbitan monolaurate), Tween 8 (sorbitan mono-oleate, polyoxyalkalene derivative), Span 4 (sorbitan monopalmitate), and Span 8 (sorbitan mono-oleate) were manufactured by Atlas Chemical Industries, Inc., Wilmington, Del. Preparation of the salts of the fatty acids. The sodium and potassium salts of the acids were prepared by adding sufficient sodium or potassium hydroxide to satisfy the acid equivalences. The acid values were obtained from Spector (7). The bases and fatty 1767 acids were heated in water until saponification was complete. The salts were dried in vacuum and weighed as required, dissolved in water, and filtered. Purified fatty acids were precipitated from solutions of the salts by use of.1 N HCl. The supernatant fluid was removed, and the precipitates were dissolved in chloroform. The solution was washed in a separatory funnel with water. The chloroform was evaporated in vacuum at 5 C; the fatty acids were weighed as required and were treated as described below. Test of anitibacterial activity. Procedures for testing the antibacterial activity of APF were modified from those described by Eisler and von Metz (3), because the fatty acids and their esters are insoluble in water. The saturated fatty acids and methyl esters were suspended in c% gelatin in water and were autoclaved at 15 psi for 1 to 15 min. Unsaturated fatty acids were prepared in this way and also by heating briefly in % gelatin in water over a flame. The mixtures were homogenized while warm with a Vortex mixer. The suspensions were stable at 35 C when the fatty acids did not exceed mg/ml. Upon addition to double-strength Heart Infusion Broth (HIB; Difco), the ph of the various mixtures was 7.2 to 7.5. Stock solutions of Tweens 2 and 8 were sterilized by filtration (Millipore Corp., Bedford, Mass.); those of Spans 4 and 8 were sterilized by autoclaving. To various concentrations of fatty acid-gelatin mixture in water (1.6 ml) were added 2 ml of double-strength HIB and.4 ml of a 1-5 dilution of a 24-hr broth culture of P. pestis strain Poona. The number of organisms in the inocula varied from 13 to 4 X 13 per ml. The zero-hour viable cell assays were made

2 1768 EISLER AND VON METZ J. BACTERIOL. by the immediate removal of. l-ml quantities of suspension, which were spread upon Blood Agar Base (BAB; Difco) and incubated at 28 C for 48 hr before the enumeration of colonies. Viability determinations were also made at 24 and 48 hr after inoculation. The tubes containing the organism-fatty acid mixtures and the control tubes were incubated statically at 35 to 36 C. Determination of free acids in Spanis 4 and 8. Free acids in Spans 4 and 8 were estimated by titrations with N HCl by use of a recording ph meter. Photographs. Mixtures of HIB, organisms (17/ml), and lauric acid ( mg/ml) were prepared in replicate and were incubated at 35 to 36 C. At, 1, and 2 hr,.2 ml of the mixtures were placed on # 1 coverslips and freeze-dried for 1 hr at a pressure 5 of Hg. The organisms were stained by Wayson's method (11) and photographed. Leakage of material from P. pestis treated with fatty acids. Samples (5-ml) of a 24-hr culture of avirulent P. pestis strain E.V. 76 (3.6 X 19 organisms/nil) were centrifuged at 17,3 X g for 3 min, and the supernatant fluid was discarded. The organisms were resuspended to original volume in.5 M potassium phosphate buffer with % sodium chloride (ph 7.6) and were centrifuged. The supernatant fluid was discarded, and the process was repeated. The organisms were finally suspended in either phosphates-buffered saline solution, the solution containing mg/ ml of sodium laurate, or the solution containing mg/mi of sodium oleate. Blanks consisted of solutions of soaps alone. Blank and test suspensions were incubated at 35 C for 21 hr and then were centrifuged. The ultraviolet absorption characteristics of the supernatant solutions were determined with the Beckman DK-1 spectrophotometer. Protein and nucleic acid concentrations were determined from the nomograph devised by E. Adams, which is based on the findings of Warburg and Christian (14) and which is distributed by Calbiochem, Los Angeles, Calif. RESULTS Antibacterial activity of fatty acids. Table 1 shows that the saturated fatty acids with 12, 14, and 16 carbon atoms were more inhibitory at 35 C than those with shorter or longer carbon chains. The unsaturated fatty acids with two and three double bonds were more effective than oleic acid. To determine the effects of refrigeration, replicate tubes were prepared and stored at to 4 C for 48 hr. Samples were removed at, 24, and 48 hr and were tested for viable organisms. In all cases, except with lauric acid which was active at 4 C, P. pestis remained viable, and the recovery of organisms was comparable to that of the original inoculum. When the tubes were removed from the refrigerator and incubated at 35 C for 48 hr, the antibacterial effects of the fatty acids became evident and the results were comparable with those in Table 1. The antibacterial effects of fatty TABLE 1. Effects offattty acids on Pasteurella pestis straini Poonia ini Heart Infusion Broth (HIB) at 35 C Fatty acid Caproic, C6 Caprylic, C8, C12, C14 Palmitic, C16 Stearic, C18 Oleic, C181 Linoleic, C182 Linolenic, C183 Gelatin-broth control (9)b Broth control (8)b ml opeib inlg.3 hr 24 hr 48 hr a No viable cells in.1 ml of undiluted mixture. bnumber of separate determinations; mean values. Gelatin-broth and broth controls were included in the assay of each fatty acid. acids were dependent upon the temperature at which the mixtures were held. Since commercially prepared fatty acids might contain impurities responsible for their antibacterial activity, we reprecipitated them from aqueous solutions of sodium salts. Thus, watersoluble impurities, if any, should have been removed, but not the chloroform-soluble impurities. The antibacterial activities of the reprecipitated fatty acids were comparable with those reported in Table 1. The techniques of viability assays insured some carry-over of fatty acids from test tubes to agar plates so that the continuing activity of acid was possible. To test this point, fatty acid-treated P. pestis was cultured on BAB

3 VOL. 95, 1968 ANTI-PASTEURELLA PESTIS FACTOR 1769 with 3%O human blood and on BAB with % starch. Since both blood (1) and starch (4) bind free fatty acids, they should eliminate any bacteriostatic effects of the residual fatty acids. Neither blood nor starch reversed the effects of lauric or myristic acids on P. pestis. Therefore, it was concluded that the actions of the fatty acids were bactericidal and not merely bacteriostatic. Furthermore, mice survived intraperitoneal inoculation with 2 X 13 P. pestis cells, which had been incubated at 37 C for 48 hr with mg of lauric or myristic acids per ml. The LD5 with untreated P. pestis strain Poona was 3 to 5 viable cells; therefore, it was concluded that the fatty acids were bactericidal. The effect of selected fatty acids at mg/ml on increasing concentrations of P. pestis are shown in Table 2. In these experiments, lauric acid killed 3.6 X 15 organisms/ml in 24 hr, and, in another experiment, 3.5 x 18. The apparently rapid action of lauric acid, as shown by the small number of viable cells at hr, was not observed in other experiments (Table 3). No explanation is TABLE 2. Effects offatty acids on several concentrations of Pasteurella pestis strain Poona in Heart Infusion Broth at 35 C Fatty acid Log inoculum ( mg/ml) Lo nouu Palmitic Linoleic Control 5.56b hr Log viable cells/mi 24 hr a No viable cells in.1 ml of undiluted mixture. badditional tests showed that lauric acid killed up to 3.5 X 18 organisms/ml. TABLE 3. Effects of lauric acid on several strains of Pasteurella pestis in Heart Infusion Broth (HIB) at 35 C Amt of lauric P. pestis strain acid per ml of HIB Al 122 E. V.76 G L 195 P mg hr 24 hr > hr _ a 8.15 a No viable cells in.1 ml of undiluted mixture. offered for the difference. Both myristic and palmitic acids (Table 2) were effective against 3.5 X 13 and 2.3 X 12 organisms/ml, respectively. In this experiment, linoleic acid was ineffective after 48 hr of incubation with P. pestis. Table 3 shows the effects of lauric acid on several strains of P. pestis. There were no significant differences in the susceptibility of the virulent (53, 139 L, 195 P) and avirulent (A1122, E.V. 76) strains. The anti-p. pestis activities of the sodium and potassium salts in HIB at ph 7.4 to 7.5 are shown in Table 4. The salts of lauric, myristic, and palmitic acids were about as active as the free fatty acids (Table 1). The salts of caproic, caprylic, and linoleic acids were not as active as the corresponding free fatty acids. On the other hand, sodium oleate was more active than oleic acid. The methyl esters of lauric, myristic, palmitic, and linoleic acids inhibited the growth of P. pestis only slightly (Table 5). The surfacective Tween 2 and Spans 4 and 8 inhibited growth slightly, whereas Tween 8 may have stimulated growth (Table 6). Electrometric titrations showed 25% hydrolysis in Span 4 and 3% hydrolysis in Span 8, which may account for the slight inhibition of growth as compared with the control. The overall increase in cell numbers, however, may be contrasted with values given for palmitic and oleic acids in Tables 1 and 4. Mixtures of fatty acids. Table 7 shows the effects of mixtures of pairs of the major fatty acids which were found in approximately equal concentration in mouse liver APF (2). Comparison with Table 1 shows that stearic, oleic, and caprylic acids, respectively, antagonized the

4 177 EISLER AND VON METZ J. BACTERIOL. TABLE 4. Effects of sodium and potassium salts offatty acids on growth of Pasteurella pestis strain Poona in Heart Infusion Broth (HIB) at 35 C Sodium salt Potassium salt Fatty acid Caproic Caprylic Palmitic Oleic Linoleic Controlsb Amt mg/ml hr 24 hr 48 hr Amt mzg/tll hr a No viable cells in.1 ml undiluted mixtures. b Mean of 13 determinations. Controls were included in the assay of each fatty acid. The -hr count indicates the size of the inoculum. TABLE 5. Effects of methyl esters offatty acids on Infusion Broth (HIB) at 35 C Methyl ester ( mg/ml of HIB) hr 24 hr 48 hr Laurate Myristate Palmitate Linoleate Control hr TABLE 6. Effects of surfacective agents on Inifusioni Broth (HIB) at 35 C Agent ( mg/ml of HIB) 48 hr hr 48 hr Tween 2 (lauric) 7. Tween 8 (oleic) Span 4 (palmitic) Span 8 (oleic) Control activities of myristic, linoleic, and lauric acids. On the other hand, the mixture of caproic and palmitic acids was somewhat less antagonistic. Table 8 presents similar results; oleic acid inhibited the activities of lauric, myristic, and palmitic acids. Combinations of the very active, saturated fatty acids (Table 9) resulted in killing of P. pestis. However, when the moderately active saturated palmitic and unsaturated linoleic acids were mixed, antagonism resulted. Morphological change. The appearance of strain Poona from a 24-hr growth in HIB at 28 C, as it was inoculated into fresh broth, is shown in

5 VOL. 95, 1968 ANTI-PASTEURELLA PESTIS FACTOR 1771 Fig. 1A. After incubation for 2 hr at 35 C, the control organisms appeared to be well developed (Fig. ib). Figure 1C shows the effect of treatment with lauric acid for 2 hr at 35 C. The organisms were fewer and appeared intact, although they TABLE 7. Effects of mixtures of fatty acids oit Infusion Broth (HIB) at 35 C Fatty acids (mg/ml of HIB) O hr 24 hr 48 hr Stearic Linoleic Oleic CaprYlic a Palmitic Caproic Gel control a No viable cells in.1 ml of undiluted mixture. TABLE 8. Effect of oleic acida on the bactericidal activity of other fatty acids on Pasteurella pestis strain Poona in Heart Infusion Broth (HIB) at 35 C Fatty acid Palmitic Gel control Amt rig/int.3.1 O hr 24 hr 48 hr b a Oleic acid ( mg/ml) in HIB to which the separate fatty acids were added to the final concentration indicated. b No viable cells in.1 ml of undiluted mixture. TABLE 9. Effects of mixtures of fatty acids on Infusion Broth (HIB) at 35 C Fatty acid mixture ( mg/ml of HIB each) O hr 24 hr 48 hr Palmitic + myristic 3.56 _ a Palmitic + lauric a a + myristic 1.78 a Linoleic + palmitic Linoleic + laurie a Linoleic + myristic 3.53 a 1.7 Gel control a No viable cells in.1 ml of undiluted mixture. were smaller than the controls. They resembled organisms treated with APF. Protein and nucleic acid. Ultraviolet absorption indicated little loss of intracellular material, since the suspending fluid of cells treated with laurate and oleate contained negligible protein and.14 and.1 mg of nucleic acids per ml, respectively. The supernatant solutions from mixtures of buffer and organisms alone contained negligible protein and.14 mg of nucleic acids per ml. DISCUSSION According to Nieman (8), the majority of bacteria susceptible to fatty acids are gram-positive. Relatively few gram-negative organisms have been reported to be inhibited by fatty acids. Walker (13) demonstrated the antibacterial activity of fatty acid soaps on Neisseria gonorrhoeae and N. meningitidis. Ley and Mueller (6) found that oleic or stearic acids, in low concentrations, inhibited the growth of N. gonorrhoeae. Spector (1) inhibited the growth of Escherichia coli with mixed acids from butter fat or corn oil. Pollock (9) inhibited Haemophilus pertussis with oleic, linoleic, and linolenic acids. Undoubtedly, other gram-negative organisms are susceptible. As reported here, P. pestis was inhibited by both saturated and unsaturated fatty acids. Of those investigated, the saturated acids containing less than 12, or more than 16, carbon atoms were relatively inactive. The unsaturated acids containing two or three double bonds were more active than oleic acid. These observations on the influence of carbon chain length and degree of unsaturation in relation to antibacterial activity are consistent with the findings of others in respect to fatty acids (8) and in respect to other surfacective agents (12). With the exception of the activities of the sodium and potassium salts of lauric, myristic,

6 1772 EISLER AND VON METZ J. BACTERIOL t... 5 t-l A B Downloaded from *9 5.t :.S.,M * o.p 'I 9 ::. i,:.1 S*.: on January 22, 219 by guest FIG. 1. (A) Pasteurella pestis strain Poona; the inoculum. (B) P. pestis in Heart Infusion Brotlh (HIB) after 2 hr at 35 C. (C) P. pestis in HIB with lauric acid ( mg/mi) after 2 hr at 35 C. Round distorted shapes are aggregates of lauric acid.

7 VOL. 95, 1968 ANTI-PASTEURELLA PESTIS FACTOR 1773 and palmitic acids, the activities of salts of the other fatty acids against P. pestis were slightly reduced. Furthermore, the presence of the CH3 group or sorbitan instead of H in the carboxyl radical largely abolished the activities of the acids tested. The results suggest that both the number of carbon atoms and the carboxyl group are important in the anti-p. pestis activity. On the other hand, less active fatty acids also contain this end group on their molecules. It is possible that the activity of the fatty acids is due to ions formed from completely dissociated molecules, even from salts of poorly soluble fatty acids. Probably, all of the salts are completely dissociated at the ph levels (7.2 to 7.5) used, but some are more effective bactericidal agents than others (e.g., caproate and caprylate). Photographs of lauric acid-treated organisms showed intact cells which, however, were arrested in development. The very slight concentrations of protein and nucleic acid in cell-free fluids from laurate- and oleate-treated organisms were not considered significantly different from control values obtained from cells treated with only phosphate-buffered solutions of sodium chloride. From these results, it appears that the fatty acids did not kill the cells primarily by causing leakage of protein and nucleic acids. However, the fatty acids may have caused leakage of some other vital factor which was not observed in these experiments. These findings and conclusions are not consistent with those of Hotchkiss (5), who reviewed the nature of the bactericidal action of surface agents and stated that cytolytic injury resulted in considerable loss of nitrogen and phosphorus compounds. The inhibition of the activities of myristic, linoleic, and lauric acids by stearic, oleic, and caprylic acids (Tables 7, 8) suggests an explanation for the anomaly in experiments with APF. Eisler et al. (2) found that wheat germ and pancreatic lipases inhibited the activity of extracts of APF and therefore concluded that the activity was due to lipids. Since lipases split triglycerides to free fatty acids, it was difficult, in view of the data in Tables 1 to 3, to understand why fatty acids liberated from APF would be inactive. Determinations of the fatty acid composition of chloroform extracts of APF (2) showed relatively high concentrations of stearic (14.8%) and oleic (12.1%) acids. Upon liberation from triglycerides by treatment with lipases, these fatty acids may inhibit the activity of both free and simultaneously liberated lauric, myristic, and palmitic acids. This may explain the observed inhibition of antibacterial activity by lipase, which was the basis for stating that activity resided in the lipid fraction of APF. On the other hand, we have no explanation why the stearic and oleic acids in APF do not inhibit its activity. ACKNOWLEDGMENTS This investigation was supported by the Office of Naval Research and the Bureau of Medicine and Surgery, U.S. Navy, under a contract between the Office of Naval Research and the Regents of the University of California. LITERATURE CITED 1. EGGERTH, A. H The effect of serum upon the germicidal action of soaps. J. Exptl. Med. 46: EISLER, D. M., B. HILL, E. K. VON METZ, W. CHANG, AND R. J. HECKLY Anti-Pasteurella pestis factor from mice and guinea pigs. II. Some chemical and physical characteristics. J. Immunol. 98: EISLER, D. M., ANDE. K. VON METZ Anti- Pasteurella pestis factor in the organs of normal mice and guinea pigs. I. Biological characteristics. J. Immunol. 91: :I 4. FOSTER, J. W., AND E. S. WYNN The problem of "dormancy" in bacterial spores. J. Bacteriol. 55: HOTCHKISS, R. D The nature of the bactericidal action of surface active agents. Ann. N.Y. Acad. Sci. 46: LEY, H. L., JR., AND J. H. MUELLER On the isolation from agar of an inhibitor for Neisseria gonorrhoeae. J. Bacteriol. 52: NATIONAL ACADEMY OF SCIENCE Handbook of biological data (W. S. Spector, ed.), p. 18. W. B. Saunders Co., Philadelphia. 8. NiMAN, C Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriol. Rev. 18: POLLOCK, M. R The effects of long-chain fatty acids on the growth of Haemophilus pertussis and other organisms. Symp. Soc. Exptl. Biol. 3: SPECrOR, H The comparative effect of the fatty acids of butterfat and corn oil on the growth and metabolism of microorganisms. Arch. Biochem. 11: STn1r, E. R., P. W. CLOUGH, AND S. E. BRANHAM Practical bacteriology, hematology, and parasitology, p. 366, 1th ed. The Blakiston Co., Philadelphia. 12. VALKO, E. I Surface active agents in biology and medicine. Ann. N.Y. Acad. Sci. 46: WALKER, J. E The germicidal properties of soap. J. Infect. Diseases 38: WARBURG,., AND W. CHRISTIAN Isolierung und Kristallisation des Garungs ferments. Enolase. Biochem. Z. 31: