yield, intracellularly, a toxic aglycon moiety Such a compound would be useful in the isolation of salmonellae from highly contaminated substrates

Transcription

1 APPuED MICROBIOLOGY, Sept. 1967, p Copvright 1967 American Society for Microbiology Vol. 15, No. 5 Printed In U.S.A. Selective Inhibition of Escherichia coli in the Presence of Salmonella typhimurium by Phenethyl f-d-galactopyranoside M. A. JOHNSTON AND F. S. THATCHER Diivision ofmicrobiology, Food and Drug Directorate, Department ofnational Health and Welfare, Ottaa, Ontario, Canada Received for publication 1 May 1967 Phenethyl f3-d-galactopyranoside (PEG) as hydrolyzed by the -galactosidase of Escherichia coli to form the toxic product phenethyl alcohol. Salmonella typhimurium did not hydrolyze PEG. In mixed culture, the ratio of S. typhimurium to E. coli as increased by groing the organisms in lactose broth containing 2.5% PEG. The high concentration of PEG required for inhibition of E. coli can be attributed to inadequate cell permeability rather than to prevention of f3-galactosidase induction. The ability to ferment lactose is one of the major biochemical distinctions beteen the genera Escherichia and Salmonella. Lactose is utilized by coliforms but not by salmonellae. Nevertheless, lactose has been used extensively in media for isolation of Salmonella from foods hich frequently contain large numbers of coliforms (5, 6, 9, 13). Isolation of Salmonella from dried-egg products entails a to-step enrichment: pre-enrichment in a nonselective medium folloed by selective enrichment in selenite or tetrathionate broth. Preenrichment in lactose broth as introduced by North (13), but Taylor and Silliker (18), using ducitol, mannitol, and lactose, found that lactose had no advantage over the other to sugars. Selective enrichment in selenite broth containing lactose as introduced by Leifson (9), and additional studies comparing lactose ith other carbohydrates ere carried out in several laboratories (7, 16, 17, 19, 21). To date, there is no conclusive evidence for any superiority of lactose over other sugars in either pre-enrichment or selective enrichment media. Recently, attempts have been made to separate lactose-fermenting organisms from organisms that do not ferment lactose on the basis of,3-galactosidase identification rather than on the conventional lactose fermentation (2, 11, 14). The assay procedure is based on the ability of f3-galactosidase to split f3-d-galactosides other than lactose. This ork has prompted us to investigate the possibility of selectively inhibiting,b-galactosidase-producing microorganisms by applying a f3-d-galactoside hich upon hydrolysis ould 1223 yield, intracellularly, a toxic aglycon moiety Such a compound ould be useful in the isolation of salmonellae from highly contaminated substrates by permitting normal groth of Salmonella species hile inhibiting the groth of coliforms. MATERIALS AND METHODS Test organisms. The S. typhimurium culture as isolated in this laboratory from podered egg. E. coli 15 as obtained from the Laboratory of Hygiene, Department of National Health and Welfare, Ottaa. Media and Reagents. All bacteriological media ere purchased from Difco Laboratories, ith the exception of a defined medium employed in a fe experiments to study the effect of phenethyl f,-d-galactopyranoside on the induction of,3-galactosidase. The defined medium contained.2% NH4Cl,.3% KH2PO4,.6% Na2HPO4,.3% NaCl,.8% MgCl2 6H2,.1% Na2SO4.1H2, and.5% glycerol. Phenethyl-,3-D-galactopyranoside (PEG) as obtained from Calbiochem, Los Angeles, Calif., and o-nitrophenol-,8-d-galactopyranoside (ONPG), from Sigma Chemical Co., St. Louis, Mo. Media containing galactosides ere sterilized by filtration. Cultural methods and cell preparations. Cell suspensions ere obtained by groing the organisms in Nutrient Broth for 18 to 24 hr at 35 C. Cells used for inoculation into the defined medium ere ashed tice ith phosphate buffer (3). Dried cells ith 86% viability ere obtained by freeze-drying ashed cells in a menstruum composed of 2% Casitone, 1% glutamate, 4% sucrose, and.2% gelatin. Cell-free extracts ere prepared by disrupting ashed cells in a 1-kc Raytheon sonic oscillator operated at 25 for 2 to 25 min at to 5 C. Total viable cells ere enumerated on Plate Count Agar; E. coli, on MacConkey Agar, and S. typhimurium, on Brilliant Green Sulpha Agar. Assay of,3-galactosidase. Enzyme activity as

2 1224 JOHNSTON AND THATCHER APPL. MICROBIOL. assayed according to the method of Lederberg (8) ith the use of cells gron for 18 to 2 hr. Suitable amounts of cell suspension or cell-free extract ere diluted to 9. ml ith.1 M sodium phosphate buffer (ph 7.5). The samples ere equilibrated in a ater bath at 35 C for 1 min, and 1 ml of.5 M ONPG as added. After an additional 2 min, the reaction as stopped by the addition of 1 ml of 1. M sodium carbonate. Samples ere centrifuged at 3,2 X g to remove any precipitate, and optical densities ere measured at 42 m,u in a Bausch & Lomb model 2 colorimeter. One unit of -galactosidase is defined as the amount of enzyme hich liberates 1 m,umole of o-nitrophenol (ONP) per min. The specific activity is defined as units per milligram (dry eight) of hole cells. Induction of,3-galactosidase by PEG. The effect of PEG on the induction of,3-galactosidase in E. coli as studied in the chemically defined medium. As inoculum, cells ere gron for 24 hr in the same medium ithout PEG. When preinduced cells ere required for inoculation,.5%o lactose as added to the defined medium. Inhibition ofgroth ofe. coli by PEG. Lactose broth containing PEG as inoculated ith a ashed suspension of cells hich had been gron in lactose broth. At appropriate intervals, the total viable population as enumerated. Partial purification of,3-galactosidase. A crude preparation of,3-galactosidase from E. coli as obtained from cells gron in lactose broth at 35 C for 24 hr. The cells ere harvested by centrifugation, ashed tice in.1 M potassium phosphate buffer (ph 7.), and disrupted by sonic oscillation. Cell debris as removed by centrifugation at 1, X g for 25 min. The enzyme as precipitated from the cell-extract at to 5 C by adding solid ammonium sulfate to 8% of saturation. The precipitate as obtained by centrifugation, redissolved in phosphate buffer (ph 7.), and dialyzed against 5 volumes of the same buffer at to 5 C for 18 hr. Nucleic acids ere precipitated by the addition of protamine sulfate to a concentration of 1 mg/ml. The treatment as repeated if more than 1% of the original nucleic acid content remained, as determined by ultraviolet absorption (2). Further purification as accomplished by precipitation from a 4% saturated solution of ammonium sulfate. Modification of cell permeability. Washed-cell suspensions ere incubated at 35 C in the presence of 2 X 1- M ethylenediaminetetraacetate (EDTA; 1) for 2 min or in.65 M dimethylsulfoixde (DMSO; 4) for 12 min. A.9-ml amount of PEG lactose medium as added directly to.1 ml of the EDTA-treated cell suspension, hereas the cells treated ith DMSO ere separated from the DMSO by centrifugation and resuspended in 1 ml of the medium. RESULTS Groth in mixed culture. Groth curves of S. typhimurium and E. coli in lactose broth are shon in Fig. 1. Mixed cultures containing equal numbers of each species ere compared ith O 4 S S. typhimurium F1G. 1.Groth Es coli typhimuriuminlactose at35. Ophsmbols, r 4 it FIG. 1. Groth of Escherichia coi and Salmonella typhimurium in lactose broth at 35 C. Open symbols, inoculum in ratio of 1: 1; solid symbols, inoculum in ratio of 1.4 X 16: 1. mixed cultures containing large numbers of E. coli and lo numbers of S. typhimurium. Groth of both organisms as almost identical hen the inoculum as approximately the same. Hoever, hen the initial numbers of E. coli cells greatly exceeded those of S. typhimurium, groth of the latter as suppressed during the first 24 hr. Continued incubation beyond 24 hr resulted in attainment of approximately equal numbers of both organisms. Results similar to those shon in Fig. 1 ere also obtained hen lactose as omitted from the medium. 3-Galactosidase activity. /3-Galactosidase as assayed in three types of preparations, all derived from equal numbers of cells gron in lactose broth for 24 hr. These ere: (i) ashed cells, (ii) dried cells, and (iii) cell-free extracts produced by sonic oscillation. S. typhimurium did not contain,3 galactosidase, but the enzyme as present in E. coli. The specific activities of ashed, dried, and sonically treated cells ere 32, 86, and 3,9, respectively. The induction time of f-galactosidase in E. coli as determined in lactose broth. Figure 2 shos that cultures inoculated ith resting cells became more rapidly than those inoculated ith dried cells. Apparently this as due to the rapidity of groth. Although both cultures ere inoculated to obtain 17 viable cells/ml, the dried cells required approximately three times as long to

3 VOL. 15, 1967 SELECTIVE INHIBITION OF E. COLI I_ 7-INOCULUM RESTING CELLS % DRIED CELLS > 1- C) 2 9 U- 'L 15I_ In Lo )~ < 5 I/ / /-ONE GENERATION-/ / MINUTES FIG. 2. Induction of f3-galactosidase in normal and dried cells ofescherichia coli U FIG.3.1% 6 5- )l q 1[ % > -~~~~~~~~~1.5 % OO % FIG. 3. Effect of concentration of phenethyl 63-Dgalactopyranoside on the groth of Escherichia coli in lactose broth. Washed-cell suspension of a 24-hr Nutrient Broth culture as added to give an initial concentration of 55 cells per ml. enter the logarithmic phase of groth. Hoever, after each culture had undergone one generation, the specific activities ere identical. Inhibition ofgroth by PEG. Since.25 % phenethyl alcohol (PEA) prevents the groth of E. coli in lactose broth (1), concentrations of the same magnitude ere investigated ith PEG. Figure 3 shos the effect of PEG on groth of E. coli. With increasing concentrations of PEG, the lag phase as prolonged, and the final number of viable cells as reduced, hereas the groth rate during the logarithmic phase as not affected. a. z C.) -J -i H U R S FIG. 4. Effect of 2.5% phenethyl #-D-galactopyranoside on mixed cultures of Escherichia coli and Salmonella typhimurium. After 24 and 72 hr of incubation, cultures ith 2.5% PEG contained, respectively, 1 and.1 % of the viable cells found in cultures ithout PEG. Additional incubation up to 12 hr did not change the latter ratio. The effect of 2.5% PEG as tested on a mixed culture inoculated ith E. coli and S. typhimurium in a ratio of 1:1. E. coli (Fig. 4) as inhibited to approximately the same extent as shon in Fig. 3, but S. typhimurium as only slightly inhibited. This slight inhibition of S. typhimurium, probably the result of phenethyl alcohol release by E. coli. as not obtained hen S. typhimurium as gron in pure culture in broth containing 2.5% PEG. When the ratio of E. coli to S. typhimurium (E/S ratio) as increased to 23,9:1 in lactose broth, 2.5% PEG gave a selective advantage to groth of S. typhimurium (Table 1). Within 5 hr, in the absence of PEG, the E/S ratio increased over 2-fold; in contrast, in the presence of 2.5% PEG, the E/S ratio decreased to less than one-half that obtained at the beginning of the experiment. After additional incubation up to 24 hr, the culture ith 2.5% PEG still contained a much loer E/S ratio than the culture ithout PEG. Induction of,f-galactosidase. The use of PEG for selective inhibition of E. coli requires information concerning the effect of PEG and its aglycon moiety, PEA, on induction of,b-galactosidase. Some galactosides inhibit induction (12). To obviate unknon effects of lactose broth on

4 1226 JOHNSTON AND THATCHER APPL. MICROBIOL. TABLE 1. Effect of 2.5% phenethyl 3-D-galactopyranoside on a mixed culture of Escherichia coli and Salmonella typhimurium in lactose broth Time % PEG 2.5% PEG (br) E. coli S. typhimurium E/S' ratio E. coli S. typhimurium 715, 3 23,9 715, ,, 1 56, 3,6, ,, 2,, 35 5,6, 16,, a E = E. coli; S = S. typhimurium. TABLE 2. Induction ofol-galactosidase in Escherichia coli by phenethyl O3-D-galactopyranoside (PEG) Compound tested (1-a M))a O-Galactosidaseb (specific activity) Noninduced inoculumc Preinduced inoculumc None PEG ,18 Lactose... 2,72 3, 55 PEA-lactose ,25 a At this concentration, PEA and PEG did not affect groth. b The assay as performed after 5 hr of incubation in the defined medium at 35 C. c Initial cell concentration as 16 per ml. induction, e used the defined medium containing mineral salts and glycerol. Table 2 shos that, in the defined medium, PEG is able to induce 3-galactosidase, but it is only one-third as efficient as lactose. PEA interfered slightly ith induction by lactose hen the inoculum as preinduced but inhibited almost completely the induction by lactose hen the inoculum as not preinduced. Similar inhibition by PEA has been shon by Rosenksanz et al. (15). When an undefined medium (lactose broth) replaced the defined medium, noninduced inocula TABLE 3. gre as ell as induced inocula in the presence of 2.5% PEG. Although the amount of groth as only about 3 to 4% of that obtained in the absence of PEG, the specific activities ere identical. Thus, the PEA formed, although it inhibited groth, did not inhibit synthesis of f-galactosidase. Permeability. Some galactosides do not enter bacterial cells readily, but permeability of the cell can be increased by treatment ith EDTA (1). Table 3 shos that treatment of cells ith 2 X 1-4 M EDTA prior to addition of 2.5% PEG increased the inhibitory effect of PEG on groth of E. coli. The 2-min pretreatment ith EDTA increased the toxicity of PEG during 1 hr of incubation, as indicated by a reduction in cell numbers from 4.4 x 16 per ml to.6 X 16 per ml. During the same period, the pretreated cells gre as ell as untreated cells in lactose broth ithout PEG. Similar results ere obtained ith DMSO. Cells hich ere shaken in lactose broth containing.65 M DMSO for 12 min before being added to the 2.5% PEG lactose medium decreased in viable numbers from 6. X 16 to 3.6 X 16 during 1 hr of incubation. The effect of DMSO on cell permeability as not as significant as EDTA. As a further indication that the relative impermeability of E. coli limited the potential lethality of PEG, e added partially purified Effect of pretreatment ith EDTA and DMSO on groth of Escherichia coli in 2.5% PEG-lactose medium Viable cells per ml Type of treatment Lactose broth for 1 hr at 35 C After pretreatment Ratio, ith With PEG Without PEG PEG/ithout PEG EDTA absent X X X EDTA present X 16.6 X X 16.8 DMSO absent X X X DMSO present X X X 16.33

5 VOL. 15, 1967 SELECTIVE INHIBITION OF E. COLI 1227,B-galactosidase to cultures in lactose broth containing 2.5% PEG. The partially purified enzyme (7.2 units per ml) produced sufficient PEA to decrease the viable cell count of noninduced E. coli from 7 x 15 to 2.2 X 15 after 5 hr of incubation. In contrast, during the same period preinduced cells ithout the partially purified enzyme increased from 75 x 15 to 8 x 15. These observations further emphasize that PEG does not enter the cell readily. DISCUSSION An examination of the groth curves of S. typhimurium and E. coli has shon that, ith the particular media tested, both organisms demonstrate similar rates of multiplication under comparable conditions of groth. This as observed in mixed culture (1:1 ratio) ith actively groing cells. When S. typhimurium as numerically inferior at the outset, S. typhimurium underent a prolonged lag period, but both species approached equality in numbers after 36 to 48 hr. The addition of lactose to nutrient broth did not significantly affect the relative groth of the to organisms. Thus, lactose as of no value in preenrichment under the conditions tested. If pre-enrichment of dried bacteria in foods such as egg poder could be undertaken in a lactose broth containing PEG or a similar galactoside, some selective enrichment of salmonellae might be obtained in this medium. For this reason, e have investigated the ability of dried E. coli to synthesize 3-galactosidase. We found (Fig. 2) that, although a longer time as required for fb-galactosidase induction in cultures inoculated ith dried cells than ith resting cells, the enzyme activity from each type of preparation shoed a direct relationship to viable-cell count. This ork has shon that the enzyme in lactose-fermenting organisms may be used to hydrolyze f3-d-galactosides containing a toxic moiety, ith the intracellular liberation of the toxic product, e.g., hydrolysis of PEG to yield PEA. As a result, the ratio of E. coli to S. typhimurium in mixed culture is altered in favor of S. typhimurium, hich does not have the enzyme to hydrolyze PEG. We found normal groth of S. typhimurium and a reduction of E. coli numbers after a 24-hr incubation in the presence of PEG. Hoever, the concentration of PEG required for this effect as higher than that hich ould provide a comparable toxic amount of PEA, assuming complete hydrolysis of the PEG. The lo level of toxicity obtained ith PEG does not appear to be due to inhibition of f3- galactosidase synthesis, because PEG as shon to be an inducer of the enzyme, rather than an inhibitor. Also, studies ith partially purified enzyme system from E. coli shoed that PEG is readily attacked by 3-galactosidase. Furthermore, by increasing the permeability of E. coli to PEG, through the use of such agent, as EDTA and DMSO, it has been possible to demonstrate an increase in toxicity. These observations indicate that PEG is not readily available to 3-galactosidase ithin the cells of E. coli. Hoever, it is possible that PEGlactose broth ould have some advantage over lactose broth if the incubation period ere extended to at least 24 hr. Unfortunately, the high cost of PEG precludes extensive use of this compound at present. The principle of selective action by use of a substrate that gives rise, intracellularly, to a substance that is toxic only to organisms possessing a specific enzyme appears to sho promise. Presently e are investigating other 3-D-galactosides hich may have greater ability to penetrate the cell and hich may be more toxic. ACKNOWLEDGMENTS We gratefully acknoledge the technical assistance received from Robert Gagne and the helpful suggestions offered by H. Pivnick and A. H. W. Hauschild during the preparation of the manuscript. LITERATURE CITED 1. BERRAH, G., AND W. A. KONETZKA Selective and reversible inhibition of the synthesis of bacterial deoxyribonucleic acid by phenethyl alcohol. J. Bacteriol. 83: BULLOW, P The ONPG test in diagnostic bacteriology. 2. Comparison of the ONPG test and the conventional lactose-fermentation test Acta Pathol. Microbiol. Scand. 6: BUTTERFIELD, C. T The selection of a dilution ater for bacteriological examinations. J. Bacteriol. 23: FOWLER, A. V., AND I. ZABIN Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92: GEORGALA, D. L., AND M. BOOTHROYD A system for detecting salmonellae in meat and meat products. J. Appl. Bacteriol. 28: HALL, H. E., D. F. BROWN, AND R. ANGELOTTI The quantification of salmonellae in foods by using the lactose pre-enrichment method of North. J. Milk Food Technol. 27: HOBBS, B. C., AND V. D. ALLISON Studies on the isolation of Bact. typhosum and Bact. paratyphosum B. Monthly Bull. Min. Health Lab Serv. 4: LEDERBERG, J The beta-d-galactosidase of Escherichia coli, strain K-12. J. Bacteriol. 6: LEIFSON, E Ne selenite enrichment media

6 1228 JOHNSTON AlNED THATCHER APPL. MICROBIOL. for the isolation of typhoid and paratyphoid (Salmonella) bacilli. Am. J. Hyg. 24: LEIVE, L A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc. Natl. Acad. Sci. U. S. 53: LE MINOR, L., AND F. BENHAMEDA Avantages de la recherche de la j8-galactosidase sur celle de la fermentation du lactose en milieu complexe dans le diagnostic bacteriologique, en particulier des Enterobacteriaceae. Ann. Inst. Pasteur 12: MULLER-HILL, B., H. V. RICKENBERG, AND K. WALLENFELS Specificity of the induction of the enzymes of the lac operon in Escherichia coli. J. Mol. Biol. 1: NORTH, W. R Lactose pre-enrichment method for isolation of Salmonella from dried egg albumen. Appl. Microbiol 9: PIcKETr, M. J., AND R. E. GOODMAN Galactosidase for distinguishing beteen Citrobacter and Salmonella. Appl. Microbiol. 14: ROSENKRANZ, H. S., H. S. CARR, AND H. M. Ross Phenethyl alcohol and messenger RNA. Biochem. Biophys. Res. Commun. 17: SMITH, H. G Observations on the isolation of salmonellae from selenite broth. J. Appl. Bacteriol. 22: STOKES, J. L., AND W. W. OSBORNE A selenite brilliant green medium for the isolation of Salmonella. Appl. Microbiol. 3: TAYLOR, W. I., AND J. H. SILLIKER Isolation of salmonellae from food samples. IV. The comparison of methods of enrichment. Appl. Microbiol. 9: TAYLOR, W. I., J. H. SILL1KER, AND H. P. ANDREWS Isolation of salmonellae from food samples. I. Factors affecting the choice of media for the detection and enumeration of Salmonella. Appl. Microbiol. 6: WARBURG, O., AND W. CHRISTIAN Isoliering und Krystallization des Garungsferments enolase. Biochem. Z. 31: WILLIAMS SMITH, H The evaluation of culture media for the isolation of salmonellae from faeces. J. Hyg. 5: Donloaded from on November 1, 218 by guest